Methods of 6-0 sulfating polysaccharides and 6-0 sulfated polysaccharide preparations

ABSTRACT

Disclosed are methods of 6-O-sulfating glucosaminyl N-acetylglucosamine residues (GlcNAc) in a polysaccharide preparation and methods of converting anticoagulant-inactive heparan sulfate to anticoagulant-active heparan sulfate and substantially pure polysaccharide preparations may by such methods. Also disclosed is a mutant CHO cell which hyper-produces anticoagulant-active heparan sulfate. Methods for elucidating the sequence of activity of enzymes in a biosynthetic pathway are provided.

RELATED APPLICATIONS

[0001] Benefit of priority is claimed to U.S. Provisional ApplicationSerial No. 60/279,523, which was filed on Mar. 28, 2001, and U.S.Provisional Application Serial No. 60/316,289, which was filed on Aug.30, 2001, the disclosures of which are herein incorporated by reference.

GOVERNMENT SUPPORT

[0002] Work described herein was supported by National Institutes ofHealth Grants 5-P01-HL41484, 5-R01-HL58479, and GM-50573. The Governmenthas certain rights in the invention.

FIELD OF THE INVENTION

[0003] The present invention relates generally to the biosynthesis ofglucosaminoglycans, and in particular to 6-O-sulfating polysaccharides.

BACKGROUND

[0004] Heparin/heparan sulfate (HS) is a linear polymer covalentlyattached to the protein cores of proteoglycans, which are abundant andubiquitously expressed in almost all animal cells. HS is assembled bythe action of a large family of enzymes that catalyze the followingseries of reactions: chain polymerization comprising the alternatingaddition of N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcUA)residues; GlcNAc N-deacetylation and N-sulfation; glucuronic acidepimerization to L-iduronic acid (IdoUA); 2-O-sulfation of uronic acidresidues; and 3-O- and 6-O-sulfation of glucosaminyl residues.

[0005] The interaction between HS and various proteins occur in highlysulfated regions of the HS. Furthermore, the specificity of anyHS-protein interaction is largely dictated by arrangement of sulfatesalong the HS chain. For example, the pentasaccharide sequence,GlcNAc/NS6S—GlcUA—GlcNS3S±6S—IdoUA2S—GlcNS6S, represents the minimumsequence required for antithrombin (AT) binding, where the 3S(3-O-sulfate) and 6S (6-O-sulfate) groups constitute the most criticalelements involved in the binding (12-16). The AT-HS complex has potentanticoagulant properties. Several enzymes involved in anticoagulantheparan sulfate (HS^(act)) biosynthesis have been purified and cloned.For example, glucosaminyl 3-O sulfotransferase (3-OST) and glucosaminyl6-O-sulfotransferase (6-OST) proteins have been purified and cloned(17,18). Multiple isoforms of 6-OST and 3-OST proteins have beenisolated and shown to have tissue-specific expression patterns anddistinct substrate specificities.

[0006] Two different sulfated domains are present in HS, namely, the NSdomain and NAc/NS domain (40,41). The NS domains consist of contiguousiduronosyl N-sulfoglucosamine units, while the NAc/NS domain consists ofalternating N-acetylated and N-sulfated disaccharides. Acceptorspecifcities of 6-OST-1, 6-OST-2, and 6-OST-3 using N-sulfated heparosanand desulfated re-N-sulfated heparin as substrate, indicated that thesulfation of position 6 of the N-sulfoglucosamine residues in the NSdomain is catalyzed by 6-OST-1, 2A, 2B, and 3 and the sulfation ofposition 6 of the N-sulfoglucosamine residues in the NA/NS domain arecatalyzed by 6-OST-2 and 6-OST-3 (2).

[0007] Tissue-specific and developmentally regulated expression of theHS biosynthetic enzymes and enzyme isoforms produce HS chains withspecific sequences (1-3). This microheterogeneity enables HS to interactwith a broad array of protein ligands that modulate a wide range ofbiological functions in development, differentiation, homeostasis, andbacterial/viral entry (reviewed in refs (4-11)). Syntheticpolysaccharides which possess such specific sequences may be used tomodulate such biological functions.

[0008] Heparin preparations, particularly preparations comprisingHS^(act), have been used clinically as anticoagulant therapeutics forthe prevention and treatment of thrombotic disease. HS^(act)preparations have also been used to maintain blood fluidity inextracoporeal or corporeal medical devices such as dialysis devices andstents, respectively.

SUMMARY OF THE INVENTION

[0009] In one aspect, the present invention features methods oftransferring a sulfate on to the 6-O position of a GlcNAc sugar residuein a polysaccharide preparation, the method comprising the steps of (a)providing a polysaccharide preparation having GlcNAc sugar residues, and(b) contacting the polysaccharide preparation with 6-OST protein in thepresence of a sulfate donor under conditions which permit the 6-OSTprotein to add a sulfate the 6-O-position of a GlcNAc sugar residue. Inpreferred embodiments the sulfate donor is PAPS.

[0010] In some embodiments, the polysaccharide preparation comprisesglucuronic acid (GlcUA) residues; GlcUA—GlcNAc sugar residues;disaccharides selected from the consisting of GlcUA/IdoUA—GlcNS,IdoUA2S—GlcNS, and GlcUA—GlcNS3S. In some preferred embodiments, thepolysaccharide preparation includes the pentasaccharide sequence of theantithrombin binding motif, namely,GlcNAc/NS6S—GlcUA—GlcNS3S±6S—IdoUA2S—GlcNS6S.

[0011] In some embodiments, the polysaccharide preparation includesprecursor saccharides for the antithrombin binding motif for example.GlcNAc/NS—GlcUA—GlcNS3S±6S—IdoUA2S—GlcNS6S, GlcNAc/NS6S—GlcUA—GlcNS3S±—IdoUA2S—GlcNS6S, GlcNAc/NS6S—GlcUA—GlcNS3S±6S—IdoUA2S—GlcNS.Particularly preferred precursors include IdoA/GlcA—GlcNAc6S,IdoA/GlcA—GlcNS6S, and IdoA2S—GlcNS6S.

[0012] In some embodiments the 6-OST protein is a recombinant proteinproduced in an expression system such as baculovirus cells, bacteriacells, mammalian cells, or yeast cells. In some preferred embodimentsthe 6-OST is human 6-OST, however, 6-OST from other mammals may also beused. In particularly preferred embodiments, the 6-OST protein comprisesa polypeptide selected from the group consisting of (a) human 6-OST-1(SEQ ID NO. 3); (b) human 6-OST-2A (SEQ ID NO. 4); (c) human 6-OST-2B(SEQ ID NO. 5); (d) human 6-OST-3 (SEQ ID NO. 6); (e) an allelic orspecies variant of any of a-d; and (f) a functional fragment of any oneof a-d.

[0013] In some embodiments, the suflation reaction mixture comprising atleast one chloride salt, and the pH is between 6.5 and 7.5. In preferredembodiments, the 6-OST is contacted with the polysaccharide preparationprotein in the presence of a sulfate donor for at least 20 minutes. Inother embodiments, the reaction proceeds over night.

[0014] In another aspect, the present invention features method ofenriching the portion of HS^(act) present in a polysaccharidepreparation comprising: (a) providing a 3-O-sulfated polysaccharidepreparation; and (b) contacting the preparation with 6-OST protein inthe presence of a sulfate donor under conditions, which permit the 6-OSTprotein to add a sulfate the 6-O-position of a GlcNAc sugar residue,wherein, step (b) occurs concurrent with or subsequent to step (a). Inpreferred embodiments, the sulfate donor is PAPS. In some embodiments,the polysaccharide preparation is derived from heparan; however, thepolysaccharides may be derived from other sources of polysaccharidesknown in the art.

[0015] In some embodiments, the 3-O-sulfated polysaccharide preparationis derived from a cell that expresses 3-OST-1, in alternativeembodiments, the 3-O-sufated polysaccharide preparation is prepared bycontacting HS^(inact) with 3-OST-1 protein (SEQ ID NO 2), allelic orspecies variant, or functional fragments of 3-OST-1.

[0016] In preferred embodiments, the percentage of HS^(act) present inthe polysaccharide preparation following step (b) is greater than 50%.In particularly preferred embodiments, the percentage of HS^(act)present in the polysaccharide preparation following step (b) is greaterthan 70%.

[0017] Preferred polysaccharide preparations for use in the methods ofthe invention comprise N-acetylglucosamine (GlcNAc) and glucuronic acid(GlcUA) residues. Particularly preferred polysaccharide preparations foruse in the methods of the invention comprise GlcUA/IdoUA-GlcNS,GlcUA—GlcNAc, IdoUA2S—GlcNS, and GlcUA—GlcNS3S.

[0018] In another aspect, the present invention features, a mutant CHOcell (“hyper-producer”) that produces between 28% and 50% HS^(act). Inpreferred embodiment, the hyper-producer produces 50% HS^(act) relativeto total HS produced by the cell. The mutant CHO cell may be produced bya method comprising: (a) transforming a CHO cell with multiple copies of3-OST-1, allelic or species variant or functional fragment thereof; (b)mutagenizing the cell obtained in step (a); (c) isolating a mutant cellfrom step (b) which fails to product HS^(act); and (d) transforming thecell obtained in step (c) with 6-OST. In particularly preferredembodiments, the 6-OST protein comprises a polypeptide selected from thegroup consisting of (a) human 6-OST-1 (SEQ ID NO. 3); (b) human 6-OST-2A(SEQ ID NO. 4); (c) human 6-OST-2B (SEQ ID NO. 5); (d) human 6-OST-3(SEQ ID NO. 6); (e) an allelic or species variant of any of a-d; and (f)a functional fragment of any one of a-d.

[0019] In another aspect, the present invention features, a method ofelucidating the sequence of components in a biosynthetic pathwaycomprising the steps of: (a) providing a target cell which expresses atleast the upstream components of the biosynthetic pathway; (b)transforming the target cell with multiple copies of an isolatedbiosynthetic pathway downstream gene; (c) mutagenizing the transformedtarget cell; and (d) identifying transformed and mutagenized targetcells that fail to express the phenotype characteristic of thebiosynthetic pathway. In some embodiments, that method further comprisesthe step of (e) correcting the step (d) cells. In such embodiments, thecorrecting step may comprise inserting an upstream gene into the cellsof step (d). The upstream gene may be a cDNA, genomic DNA, or afunctional fragment thereof. In preferred embodiments, the cells of step(d) are transformed with a pool of preselected cDNAs for components ofthe biosynthetic pathway, for example, a cDNA library derived from acell that expresses the characteristic non-mutant phenotype.

[0020] In some embodiments, the correcting step may comprise contactingthe cells of step (d) with the gene product of an upstream gene. Inalternative embodiments, the correcting step may comprise contacting thecells of step (d) with the mRNA, cDNA, genomic DNA, or a functionalfragment thereof for the upstream gene.

[0021] In some embodiments, the method further comprises the step ofisolating the cells from step (d), analyzing the cells of step (d),and/or isolating the upstream gene in the biosynthetic pathway.

[0022] In some embodiments, the mutagenesis step comprises a mutagenesistechnique selected from the group consisting of chemical mutagenesis ionradiation, and ultraviolet radiation. The step of identifying the genecDNA may comprise complementation analysis, Northern blot analysis,Southern blot analysis, and/or Western blot analysis. In preferredembodiments, upstream gene may be isolated using PCR or any othertechnique known in the art.

[0023] In another aspect, the present invention features methods ofreducing thrombin activity in a medical device comprising the step ofcoating the medical device with any of the substantially purepreparations and/or preparations enriched for HS^(act) disclosed herein.In preferred embodiments the medical device is an extracorporeal orintracorporeal device that contacts blood.

[0024] These and other objects, along with advantages and features ofthe invention disclosed herein, will be made more apparent from thedescription, drawings, and claims that follow.

DESCRIPTION OF THE DRAWINGS

[0025]FIG. 1 depicts IPRP-HPLC of [³⁵S]sulfate metabolic labeled HSdisaccharides. The IPRP-HPLC was performed as follows. [³⁵S]sulfatemetabolically labeled HS from parental wild-type, mutant, and correctantwere isolated and digested with a mixture of heparitinases. Theresulting disaccharides were separated on a Bio—Gel P2 column and werethen further resolved by IPRP-HPLC with appropriate internalstandards. 1. ΔUA—GlcNS; 2. ΔUA—GlcNAc6S; 3. ΔUA-GlcNS6S; 4.ΔUA2S—GlcNS; 5. ΔUA2S—GlcNS6S. Blue tracer, mutant; red tracer,correctant; black broken tracer, wild-type. The broken line indicatesthe gradient of acetonitrile.

[0026]FIG. 2 depicts IPRP-HPLC of 6-OST-1 and [³⁵S]PAPS-labeled HSdisaccharides. The IPRP-HPLC was performed as follows. Cold HS from3-OST-1 expressing CHO wild-type and precursor mutant were in vitrolabeled with purified baculovirus expressed 6-OST-1 in the presence[³⁵S]PAPS for 20 min. (A) or overnight (B). HS[³⁵S] was isolated anddigested with a mixture of heparitinases. The resulting disaccharideswere separated on a Bio—Gel P2 column and further resolved by IPRP-HPLCwith internal standards. 1. ΔUA—GlcNAc6S; 2. ΔUA-GlcNS6S; 3.ΔUA2S—GlcNS6S. Solid tracer, mutant; broken tracer, wild-type. Thebroken line indicates the gradient of acetonitrile.

[0027]FIG. 3 depicts Bio—Gel P6 fractionation of digested HS. TheBio—Gel P6 fractionation was performed as follows. 6-O-[³⁵S]sulfatetagged [³H]HS from mutant were digested with 1 mU heparitinase I for 1hour. HS^(act) oligosaccharides were obtained by AT-affinitychromatography. HS^(act) oligosaccharides were then treated with low pHnitrous acid and then NABH₄ reduced, or treated with heparitinase I, II,and heparinase were analyzed by Bio—Gel P6 chromatography.

[0028] The fractions indicated were pooled for further analysis. A,6-O-[³⁵S]sulfate tagged mutant HS^(act) oligosaccharides; B,6-O-[³⁵S]sulfate tagged mutant HS^(act) oligosaccharides treated withlow pH nitrous acid and NaBH₄; C, 6-O-[³⁵S]sulfate tagged mutantHS^(act) oligosaccharides digested with heparitinases. n=the number ofmonosaccharide units in each peak.

[0029]FIG. 4 depicts IPRP-HPLC of 6-O-sulfate tagged HS^(act) di- andtetrasaccharides. The IPRP-HPLC was performed as follows. In vitro6-O-sulfated and AT-affinity purified [³H]HS^(act) oligosaccharides weredigested with a mixture of heparitinases. The resulting di- andtetrasaccharides were separated on a Bio—Gel P6 column (FIG. 3C). (A),tetrasaccharides collected from FIG. 3C, peak 1:ΔUA—GlcNAc6³⁵S—GlcUA—GlcNS3S, peak 2: ΔUA-GlcNAc6³⁵S—GlcUA—GlcNS3S6³⁵S;(B), disaccharides of the digested tetrasaccharides in the presence ofHIP peptide; peak 1: ΔUA—GlcNAc6³⁵S, peak 2: ΔUA—GlcNS3S6³⁵S; (C),disaccharides collected from FIG. 3C, peak 1: ΔUA—GlcNS6³⁵S, peak 2:ΔUA2S—GlcNS6³⁵S. The broken line indicates the gradient of acetonitrile.

[0030]FIG. 5 depicts dual-color fluorescence flow cytometric analysis ofAT (A, C, E, and G) and FGF-2 (B. D, F, and H) binding to wild-type,mutant, and 6-OST-1 correctant. CHO wild-type (A and B); wild-type CHOcell clone with 3 copies of 3-OST-1, (C and D), mutant cell clone with 3copies of 3-OST-1 (E and F), and 6-OST-1 correctant of the mutant (G andH) were double-labeled with fluorescein-AT (A, C, E, and G) and Alexa594-FGF-2 (B, D, F, and H) and subjected to dual-color FACS.

[0031]FIG. 6 depicts HPLC anion-exchange chromatography of GAGs. TheHPLC anion-exchange was performed as follows. [³H]GlcN-Labeled GAGchains from wild-type and mutant were isolated by protease digestion andβ-elimination. Samples were analyzed by HPLC anion-exchangechromatography. Solid tracer, mutant; broken tracer, wild-type. Thebroken line indicates the concentration gradient of sodium chloride.

[0032]FIG. 7 illustrates one embodiment of a method for elucidating theHS^(act) biosynthetic pathway. In this embodiment, using recombinantretroviral transduction, the human heparan sulfate (HS)3-O-sulfotransferase 1 (3-OST-1) gene was transduced into Chinesehamster ovary (CHO) cells. 3-OST-1 expression gives rise to CHO cellswith the ability to produce anticoagulant HS (HS^(act)). A cell linecontaining 3 copies of 3-OST-1 was chosen by Southern analysis. Afterchemical mutagenesis of this cell line, FGF-2 binding positive and ATbinding negative mutant cells were FACS sorted and cloned. The advantageof having 3 copies of 3-OST-1 is that upstream genes that areresponsible for generating specific HS precursor structures can besought after chemical mutagenesis without being concerned with the lossof 3-OST-1. FGF-2 selection is employed to make certain that the mutantcells still make HS.

[0033]FIG. 8 depicts ΔUA—GlcNS3 S disaccharide structure asdetermination by capillary IPRP-BPLC coupled with mass spectrometry. TheIPRP-HPLC-MS analysis was performed as follows. Cold HS chain fromwild-type CHO cells were labeled with 3-OST-1 plus PAP³⁴S. Purified HSwas digested with a combination of 1 mU of each heparitinase I,heparitinase U, heparitinase IV, and heparinase in the presence of 0.5mg/ml heparin/heparan sulfate interacting protein (HIP) peptide. 0.5 μgof digested HS was injected into capillary IPRP-HPLC coupled with MS.Panel A, UV tracer of capillary IPRP-HPLC from 35.85 to 39.71 min., peakB contains both ΔUA-GlcNS6S and ΔUA—GlcNS3 S, and peak D containsΔUA2S—GlcNS; panel B, negative polarity MS spectra from 37.44 to 38.17min.; which equals UV peal( from 36.64 to 37.37 min.; panel C,amplification of m/z 494.0 to 501.0 region from panel B; panel D,negative polarity MS spectra from 38.17 to 39.06 min.; which equals UVpeak from 37.37 to 38.26 min.; panel E, amplification of m/z 494.0 to501.0 region from panel D.

DETAILED DESCRIPTION

[0034] Before proceeding further with a detailed description of thecurrently preferred embodiments of the instant invention, an explanationof certain terms and phrases will be provided. Accordingly, it isunderstood that each of the terms set forth is defined herein at leastas follows:

[0035] Anticoagulant heparan sulfate (HS^(act)). As used herein the term“anticoagulant heparan sulfate” or the abbreviation “HS^(act)” means asulfated HS comprising the pentasaccharide binding site forantithrombin, namely, GlcNAc/NS6S—GlcUA—GlcNS3S±6S—IdoUA2S—GlcNS6S.HS^(act) may be purified from a pool of polysaccharides by any meansknown in the art, for example, AT-affinity chromatography. Theanticoagulant activity of a sample may be quantitated using thetechniques disclosed herein, or alternatively using an assay known inthe art, for example, the Coatest Heparin assay manufactured byChromogenix, Milan, Italy.

[0036] Anticoagulant-inactive heparan sulfate (HS^(inact)) As usedherein the term “anticoagulant-inactive heparan sulfate” or theabbreviation “HS^(inact)” means a sulfated HS lacking thepentasaccharide binding site for antithrombin, namely,GlcNAc/NS6S—GlcUA—GlcNS3S±6S—IdoUA2S—GlcNS6S. Anticoagulant-inactiveheparan sulfate may also be identified and quantitated using thetechniques disclosed herein or any assay known in the art, for example,the Coatest Heparin assay manufactured by Chromogenix, Milan, Italy.

[0037] Enriched. As used herein with regard to particular polysaccharidestructures within a polysaccharide preparation, the term “enriched”means that the proportion of the polysaccharide structure in apolysaccharide preparation is statistically significantly greater thanthe proportion of the polysaccharide structure in naturally-occurring,untreated polysaccharide preparation. The polysaccharide preparations ofthe invention are enriched for 6-OST-1-sulfated polysaccharides orHS^(act) approximately 10-100 fold. For example, whereas the percentageof 6-OST-sulfated polysaccharide in a typical, unenriched preparation isbetween 0%-3%, the percentage of 6-OST-sulfated polysaccharide in theenriched polysaccharide preparations of the invention is betweenapproximately 5-9%. Likewise, whereas the percentage of HS^(act) in atypical, produced by cells culture in vitro is between approximately0-1%, the percentage of HS^(act) in the enriched polysaccharidepreparations of the invention derived from the hyper-producing mutantCHO cell of the invention is between approximately 28-50%.

[0038] Heparan sulfate. As used herein, the term “heparan sulfate” orthe abbreviation “HS” means a polysaccharide made up of repeateddisaccharide units D-glucuronic acid or L-iduronic acid linked toN-acetyl or N-sulfated D-glucosamine. The polysaccharide is modified toa variable extent by sulfation of the 2-O-position of GlcA and IdoAresidues, and the 6-O- and 3-O-positions of GlcN residues andacetylation or de-acetylation of the nitrogen of GlcN residues.Therefore, this definition encompasses all of the glycosaminoglycancompounds variously referred to as heparan(s), heparan sulfate(s),heparin(s), heparin sulfate(s), heparitin(s), heparitin sulfate(s),heparanoid(s), heparosan(s). The heparan molecules may be pureglycosaminoglycans or can be linked to other molecules, including otherpolymers such as proteins, and lipids, or small molecules.

[0039] 3-O-Sulfotransferases. As used herein, the term“3-O-Sulfotransferases” refers to the family of proteins that areresponsible for the addition of sulfate groups at the 3-OH position ofglucosamine in HS. These enzymes are present as several isoformsexpressed from different genes at different levels in various tissuesand cells. The 3-OSTs act to modify HS late in its biosynthesis(reviewed by Lindahl et al., 1998) and each isoform recognizes assubstrate glucosamine residues in regions of the HS chain that havespecific, but different, prior modifications, including epimerizationand sulfation at other nearby positions (Liu et al., 1999). Thus,different 3-OSTs generate different potential protein-binding sites inHS.

[0040] 3-OST-1. As used herein, the term “3-O-sulfotransferase-1” or theabbreviation “3-OST-1” refers to the particular isoform of the6-O-Sulfotransferase family designated as “1”. 3-OST-1 is described indetail in WO 99/22005, which is herein incorporated by reference in itsentirety. As used herein 3-OST-1 may refer to the nucleic acidcomprising the 3-OST-1 gene (SEQ. ID NO. 1) or the protein (SEQ. ID NO.2). Whether the term is applied to nucleic acids or polypeptide, it isintended to embrace minimal sequences encoding functional fragments of3-OST-1. In general, a functional fragment comprises the minimumsegments required for transfer of a sulfate to the 3-Oposition of HS.Accordingly, a functional fragment may omit, for example, leadersequences that are present in full-length 3-OST-1. WO 99/22005 providesfurther guidance regarding which segments of full-length 3-OST-1 nucleicacids and polypeptides comprise functional fragments.

[0041] 6-O-Sulfotransferases. As used herein, the term“6-O-Sulfotransferases” refers to members of the family of 6-OSTs areresponsible for the addition of sulfate groups at the 6-OH! position ofglucosamine in HS. These enzymes are present as several isoformsexpressed from different genes at different levels in various tissuesand cells. As is the case with the 3-OSTs, the 6-OSTs act to modify HSlate in its biosynthesis and each isoform recognizes as substrateglucosamine residues in regions of the HS chain that have specific, butdifferent, prior modifications, including epimerization and sulfation atother nearby positions (Liu et al., 1999).

[0042] As used herein 6-OST may refer to nucleic acids or polypeptidescomprising human 6-OST-1,-2A, -2B, and -3. Whether the term is appliedto nucleic acids or polypeptide, it is intended to embrace allelic andspecies variants, as well as minimal sequences encoding functionalfragments of 6-OST. In general, a functional fragment comprises theminimum segment(s) required for transfer of a sulfate to the 6-Oposition of an HS preparation and, in particular, GlcNAC residues of HS.Accordingly, a functional fragment may omit, for example, thetransmembrane and/or leader sequences that are present in thefill-length protein.

[0043] Substantially pure. As used herein with respect to polysaccharidepreparations, the term “substantially pure” means a preparation whichcontains at least 60% (by dab weight) the polysaccharide of interest,exclusive of the weight of other intentionally included compounds.Preferably the preparation is at least 75%, more preferably at least90%, and most preferably at least 99%, by dry weight the polysaccharideof interest, exclusive of the weight of other intentionally includedcompounds. Purity can be measured by any appropriate method, e.g.,column chromatography, gel electrophoresis, amino acid compositionalanalysis or HPLC analysis. If a preparation intentionally includes twoor more different polysaccharides of the invention, a “substantiallypure” preparation means a preparation in which the total dry weight ofthe polysaccharide of the invention is at least 60% of the total dryweight, exclusive of the weight of other intentionally includedcompounds. Preferably, for such preparations containing two or morepolysaccharides of the invention, the total weight of thepolysaccharides of the invention should be at least 75%, more preferablyat least 90%, and most preferably at least 99%, of the total dry weightof the preparation, exclusive of the weight of other intentionallyincluded compounds. Thus, if the polysaccharides of the invention aremixed with one or more other compounds (e.g., diluents, detergents,excipients, salts, sugars, lipids) for purposes of administration,stability, storage, and the like, the weight of such other compounds isignored in the calculation of the purity of the preparation.Furthermore, when the polysaccharide is a proteoglycan, the proteincomponent of the proteoglycan is excluded for purposes of calculatingpurity.

[0044] Transformation. As used herein, transformation means any methodof introducing exogenous a nucleic acid into a cell including, but notlimited to, transformation, transfection, electroporation,microinjection, direct injection of naked nucleic acid,particle-mediated delivery, viral-mediated transduction or any othermeans of delivering a nucleic acid into a host cell which results intransient or stable expression of the nucleic acid or integration of thenucleic acid into the genome of the host cell or descendant thereof.

[0045] Variant. As used herein, “variant” DNA molecules are DNAmolecules containing minor changes in a native 6-OST sequence, i.e.,changes in which one or more nucleotides of a native 6-OST sequence isdeleted, added, and/or substituted, preferably while substantiallymaintaining a 6-OST biological activity. Variant DNA molecules can beproduced, for example, by standard DNA mutagenesis techniques or bychemically synthesizing the variant DNA molecule or a portion thereof.Such variants preferably do not change the reading frame of theprotein-coding region of the nucleic acid and preferably encode aprotein having no change, only a minor reduction, or an increase in6-OST biological function. Amino-acid substitutions are preferablysubstitutions of single amino-acid residues. DNA insertions arepreferably of about 1 to 10 contiguous nucleotides and deletions arepreferably of about 1 to 30 contiguous nucleotides. Insertions anddeletions are preferably insertions or deletions from an end of theprotein-coding or non-coding sequence and are preferably made inadjacent base pairs. Substitutions, deletions, insertions or anycombination thereof can be combined to arrive at a final construct.Preferably, variant nucleic acids according to the present invention are“silent” or “conservative” variants. “Silent” variants are variants of anative 6-OST sequence or a homolog thereof in which there has been asubstitution of one or more base pairs but no change in the amino-acidsequence of the polypeptide encoded by the sequence. “Conservative”variants are variants of the native 6-OST sequence or a homolog thereofin which at least one codon in the protein-coding region of the gene hasbeen changed, resulting in a conservative change in one or more aminoacid residues of the polypeptide encoded by the nucleic-acid sequence,i.e., an amino acid substitution. In all instances, variants of thenaturally-occurring 6-OST, as described above, must be tested forbiological activity as described below. Specifically, they must have theability to add a sulfate to the 6-OH position of a sugar residue in HS.

[0046] The present invention depends, in part on the discovery that (i)6-OST is limiting enzyme in the HS^(act) biosynthetic pathway when3-OST-1 is non-limiting; (ii) 6-OST can add 6-O-sulfate to GlcNAcresidues, including the critical 6-O-sulfate in the antithrombin bindingmotif of HS; and (iii) both 3-O- or 6-O-sulfation may be the final stepin HS^(act) biosynthesis. Thus, the present invention provides methodsof synthesizing oligosaccharides comprising GlcNAc6S, preparationsenriched for HS^(act), and methods of making such preparations using6-OST.

[0047] Methods of 6-O-Sulfating Polysaccharides

[0048] In one aspect, the present invention provides methods for6-O-sulfating saccharide residues within a preparation ofpolysaccharides in which the polysaccharides includes a GlcNAc sugarresidue. These methods comprise contacting a polysaccharide preparationwith 6-OST protein in the presence of a sulfate donor under conditionswhich permit the 6-OST to convert the GlcNAc sugar residue to GlcNAc6S.In particularly preferred embodiments, the 6-OST protein comprises apolypeptide selected from the group consisting of (a) human 6-OST-1 (SEQID NO. 3); (b) human 6-OST-2A (SEQ ID NO. 4); (c) human 6-OST-2B (SEQ IDNO. 5); (d) human 6-OST-3 (SEQ ID NO. 6); (e) an allelic or speciesvariant of any of a-d; and (f) a functional fragment of any one of a-d.In preferred embodiments, the sulfate donor is 3′-phospho-adenosine5′-phosphosulfate (PAPS).

[0049] In another aspect, the present invention provides methods ofproducing HS^(act) by contacting a 3-O-sulfated polysaccharidepreparation with 6-OST protein. These methods are based upon thediscovery that 6-O-sulfation can occur after 3-O-sulfation in HS^(act)biosynthesis. In particular embodiments, a GlcNAc sugar residue whichcomprises a part of an HS^(act) precursor sequence is 6-O-sulfated. Insome embodiments, the target polysaccharide comprises part of anHS^(act) precursor sequence, for example, IdoA/GlcA—GlcNAc6S,IdoA/GlcA—GlcNS6S, and IdoA2S—GlcNS6S. In some preferred embodiments,the target polysaccharide is 3-O-sulfated prior to or concurrently with6-O-sulfation.

[0050] In another aspect, the present invention also provides for meansof enriching the AT-binding fraction of a heparan sulfate pool (i.e.,increasing the portion of HS^(act)) by contacting a polysaccharidepreparation with 6-OST protein in the presence of a sulfate donor underconditions which permit the 6-OST to convert HS^(inact) to HS^(act). Inpreferred embodiments, the sulfate donor is 3′-phospho-adenosine5′-phosphosulfate (PAPS). Conversion of the HS^(act) precursor pool toHS^(act) using the methods of the invention is particularly useful inthe production of anticoagulant heparan sulfate products which haveclinical applications as therapeutics, for example, as an agent to treator prevent thrombotic disease. Anticoagulant heparan sulfate productsmay alternatively be used as agents to maintain blood flow in medicaldevices, for example, dialysis machines. In general, the preparationsenriched for HS^(act) disclosed herein may be use in any application inwhich anticoagulant HS is employed.

[0051] In yet another aspect, the present invention provides arecombinant cell line that expresses enhanced levels of HS^(act). Invitro cell cultures produce between 0% -1% HS^(act). However; thecorrected mutant (“correctantor hyper-producer”) created by transformingCHO cells multiple copies of 3-OST-1, followed mutigenization of theresultant transformant and transformation with 6-OST-1 has been shown toexpress between 28% -50% HS^(act). This represents a significantimprovement over the percentage of HS^(act) produced by any cell lineknown to applicants at the time of filing.

[0052] The 6-O-sulfated preparations and the HS^(act) produced by themethods of the invention are useful as therapeutic agents to treatand/or prevent any condition improved by administration of ananticoagulant, for example, thrombotic disease. These compositions mayalso be used to coat the surfaces of extracorporeal medical devices(e.g., dialysis tubing) or intracorporeal devices (e.g., transplants,stents or other prosthetic implants) to reduce blood clotting on thosesurfaces.

[0053] Practice of the invention will be still more fully understoodfrom the examples, which are presented herein for illustration only andshall not be construed as limiting the invention in any way.

EXAMPLE 1 6-O-sulfation of HS in vitro

[0054] 6-O-sulfation of glucosaminylglyans in vitro may be accomplishedin any manner known in the art. As a skilled artisan would recognize, a6-O-sulfation reaction requires a 6-OST protein, or functional fragmentthereof, a target polysaccharide, a sulfate donor (preferably PAPS), anda pH in the range of 6.5-7.5 (preferably a pH of about 7.0). Thus, in apreferred procedure, the reaction mixture contains 50 mM MES (pH 7.0),1% (w/v) Triton X-100, 5 mM MnCl₂, 5 mM MgCl₂, 2.5 mM CaCl₂, 0.075 mg/mlprotamine chloride, 1.5 mg/ml BSA, either metabolically labeled [³⁵S]HSor non-radioactive HS chains, cold PAPS (0.5 mM) or [³⁵S]PAPS (25 μM,2×10⁷ cpm), and 70 ng of purified baculovirus-expressed human 6-OST-1 ina final volume of 50 μl. The mixtures may be incubated either 20 minutesor overnight at 37° C., and 200 μg of chondroitin sulfate C added. HSchains are purified by phenol/chloroform extraction and anion exchangechromatography on 0.25-ml columns of DEAE-Sephacel packed in 1 mlsyringes (20). After ethanol precipitation, the pellets are washed with75% ethanol, dried briefly under vacuum, and dissolved in water forfurther analysis.

EXAMPLE 2 6-OST generate HS^(act) in vitro.

[0055] To explain the difference between 6-OST substrate specificityobserved in vivo and previously reported specifcities, 6-OST-1 wasexpressed and purified in bacteria and baculovirus. The purifiedproteins were used to sulfate HS³⁵S derived from the precursor mutantand wild-type CHO cells. Specifically, precursor mutant HS chains weretreated with baculovirus expressed, 3-OST-1 protein, 6-OST-1 protein, orboth proteins in the presence of cold PAPS. HS^(act) was isolated byAT-affinity purification and the percentage of HS^(act) was quantitated.The results are shown below in Table 1. As Table 1 shows, the yield ofHS^(act) resulting from 6-OST-1 treatment of precursor mutant HS chain(51%) was similar to that of the CHO wild-type (64%) even though6-O-sulfation is severely decreased in the precursor mutant (FIG. 1).TABLE 1 Percentage of [S³⁵]HS^(act) 3-OST-1 and Control 3-OST-1 6-OST-16-OST-1 Wild-type CHO 26% 40% 64% 70% Precursor Mutant  7% 12% 51% 64%

EXAMPLE 3 6-OST-1 sulfation generates three kinds of 6-O-containingdisaccharides in vitro

[0056] To localize where 6-OST-1 adds 6S residues along the HS chains,equal amounts of HS from 3-OST-1 expressing wild-type and precursormutant were in vitro labeled with purified baculovirus expressed 6-OST-1in the presence of [³⁵S]PAPS either for 20 minutes or overnight. Only ˜⅓as much radioactivity was incorporated into the HS derived from 3-OST-1expressing CHO cells as compared to the HS derived from the precursormutant cells. [S³⁵]HS was isolated and digested with a mixture ofheparitinases. The resulting disaccharides (accounting for ˜94% of [³⁵S]counts) were separated on a Bio—Gel P2 column and further resolved byIPRP-HPLC with appropriate internal standards (FIG. 2, mutant, solidtracer; wild-type, broken tracer). As FIG. 2 shows, 6-OST-1 not onlyadded a 6S group on GlcNS, but 6-OST-1 also 6S group on GlcNAc residuesin both the 3-OST-1 expressing CHO HS and precursor mutant HS.

[0057] As summarized below in Table 2, more ΔUA—GlcNAc6³⁵S andΔUA—GlcNS6³⁵S disaccharides were observed from reactions run overnightthan after just 20 minutes. 6-O-sulfate incorporation was 10 timeshigher from incubation with baculovirus expressed 6-OST-1 than bacteriaexpressed 6-OST-1. However, overnight labeling using bacterial 6-OST-1generated three 6-O-sulfated disaccharides in the following proportions:ΔUA—GlcNAc6³⁵S (25%), ΔUA-GlcNS6³⁵S(20%), and ΔUA2S—GlcNS6³⁵S (55%).This ratio of 6-O-sulfated disaccharides is comparable to the ratioobserved in baculovirus 6-OST-1 overnight labeled disaccharides (FIG. 2,panel B). TABLE 2 Overnight 20 Minute Incubation IncubationΔUA-GlcNAc6³⁵S 29% 18% ΔUA-GlcNS6³⁵S 18% 12% ΔUA2S-GlcNS6³⁵S 53% 70%

EXAMPLE 4 Contribution of 6-OST in generation of HS^(act)oligosaccharides.

[0058] To further locate the 6-O-sulfate addition in AT-binding HS^(act)oligosaccharides, cold mutant HS chains were treated with purifiedBaculovirus expressed 6-OST-1 with [³⁵S]PAPS overnight. Afterheparitinase I digestion, HS^(act) oligosaccharides were affinitypurified (7% of 6-O-[³⁵S]sulfate-labeled HS^(total)). The HS^(act)oligosaccharides were then treated with low pH nitrous acid that cleavesN-sulfated residues, and a combination of heparitinases that cleaves3-O-sulfate containing sugar into tetrasaccharides and all other sugarsinto disaccharides. Treated and untreated HS^(act) oligosaccharides wererun on Bio Gel P6 columns (FIG. 3). Di- and tetrasaccharides werecollected from enzyme and low pH nitrous treated samples as indicated.The tetrasaccharides resistant to a combination of heparitinases I, II,and heparinase digestion represented the 3-O-sulfate containingtetrasaccharides as reported earlier(20,33). The presence of similaramounts of tetrasaccharides from both nitrous and enzyme degradationsuggests the 3-O-containing tetrasaccharides have the structures,UA±2S—GlcNAc6³⁵ S—GlcUA—GlcNS3 S±6³⁵S. To prove this, thetetrasaccharides (FIG. 4A) collected from enzyme digestion (FIG. 3C)were further digested into disaccharides (FIG. 4B) with heparitinase Iin the presence of HIP peptide (the same method as shown in FIG. 8).IPRP-HPLC profiles of 6-O-sulfate tagged HS^(act) di- andtetrasaccharides from FIG. 3C were shown in FIG. 8. Table 3 summarizesthe 6-O-[³⁵S]sulfate-labeled disaccharide compositions calculated basedon the HPLC data (FIG. 4). TABLE 3 Percentage of [S³⁵]HS^(act) 3-OST-1and Control 3-OST-1 6-OST-1 6-OST-1 Wild-type CHO 26% 40% 64% 70%Precursor Mutant  7% 12% 51% 64%

[0059] In HS^(act) oligosaccharides, 6-OST adds 6-O-sulfates not only atGlcUA/IdoUA—GlcNS, GlcUA—GlcNAc, and IdoUA2S—GlcNS, but also atGlcUA—GlcNS3S. These results show that 6-OST is the enzyme that not onlyputs the critical 6-O-sulfate group in HS^(act) oligosaccharides, butalso other 6-O-sulfate groups in HS^(act) oligosaccharides as well.3-OST-1 and 6-OST are therefore the critical enzymes for the generationof HS^(act).

[0060] 3-OST-1, usually existing in limited amounts, acts upon HS^(act)precursor to produce HS^(act) and upon HS^(inact) precursor to produce3-O-sulfated HS^(inact) (17,19). When 3-OST-1 is no longer limiting, thecapacity for HS^(act) generation is determined by the abundance ofHS^(act) precursors (20). Since in vitro 3-O-sulfation can transformHS^(inact) into HS^(act), it was previously believed that 3-O-sulfationis the final modification step during biosynthesis of HS^(act).Surprisingly, in vitro 6-O-sulfation was also shown to transform3-O-sulfate containing HS^(inact) into HS^(act). Thus, the presentdisclosure provides methods of enriching a polysaccharide preparation ofHS^(act) by contacting a HS^(inact) with 6-O-sulfate protein and asulfate donor under conditions which permit 6-OST-1 to sulfate a GlcNAcsugar residue.

EXAMPLE 5 6-OST-1 corrected mutant makes HS, 50% of which is HS^(act).

[0061] To determine whether the diminished 6-OST activity in theprecursor mutant caused the precursor mutant's deficiency in AT binding,precursor mutant was transduced with 6-OST-1 cDNA. To create the 6-OST-1cDNA, CHO 6-OST-1 coding region was amplified and sequenced from theCHO-K1 quick-clone cDNA library by PCR. Since only partial 6-OST-1coding sequence from CHO cells has been reported (32), the complete CHO6-OST-1 sequence was deposited in Genbank (accession number: AB006180).Stable 6-OST-1 transfectants were screened by FACS. Specifically, thecells were labeled with fluorescein-AT and Alexa 594-FGF-2 and thensubjected to dual-color FACS. The FACS analysis for a correctant cell isshown in FIG. 5, at panels G and H. The correctants with high AT bindingaffinity were single-cell-cloned. HS[³⁵S] from correctants was isolatedby AT-affinity chromatography and analyzed The correctant produced HSand HS^(act). Surprisingly, approximately between 28% and 50% of totalHS produced by the correctant was HS^(act). The only cultured cell knownto the applicants at the time of the filing the instant applicationproduces approximately 0% -1% ^(HSact.)

EXAMPLE 6 Correctant ells produce a greater percentage of GlcNAc6S andGlcNS6S residues in vivo than either the wild-type CHO cells expressing3-OST-1 and the precursor mutant cells.

[0062] The disaccharide composition of the HS derived from correctantcells was shown to a comprise a greater percentage of GlcNAc6S andGlcNS6S residues than CHO cells or mutant cells as follows. HS[³⁵S] from3-OST-1 expressing CHO cells, precursor mutant cells and correctantcells was isolated and digested with a mixture of heparitinases. Theresulting disaccharides were independently separated on a Bio—Gel P2column and further resolved by IPRP-HPLC (FIG. 6). The relativepercentages are set forth below. TABLE 4 Disaccharide Wild-typePrecursor mutant Correctant GlcNAc6S 7% 5%  9% GlcNS6S 9% 4% 13%

[0063] Precursor Mutant

[0064] The present disclosure also provides a method, which constitutesa general approach for defining and obtaining components of biosyntheticpathways when (1) the gene for a downstream or terminal biosyntheticenzyme has been isolated, and (2) an assay for the downstream product(s)is available. This method of delineating biosynthetic pathways comprisesplacing multiple copies of the gene for a down-stream component of thepathway (i.e., a component acting at or near the end of a biosyntheticpathway) into a target cell line to produce a multi-expresser;mutagenizing the multi-expresser to obtain mutants deficient inup-stream component (i.e., components that generate precursor structuresearlier in the pathway than the downstream component); and analyzing theprecursor mutants. In some embodiments the method further comprises“correcting” the precursor mutant, for example, by transducing themutant with the gene encoding a putative precursor protein. Thetransduction may be accomplished using one or more previously identifiedcomponents of the biosynthetic pathway or using a “shotgun” libraryapproach. In other embodiments, the correction may entail contacting theprecursor mutant with the gene products of the biosynthetic pathway andscreening for the phenotype of the wild-type, for example, ligandbinding.

[0065] The advantage of a cell line containing multiple copies of aterminal or downstream gene product is that the activity of thedownstream gene product remains intact following mutagenesis, therefore,upstream gene products will determine the phenotype of the mutagenizedcell. Thus, the present invention provides methods of generating mutantsspecifically defective for upstream gene products, as well as, methodsfor isolating downstream components and delineating biosyntheticpathways.

EXAMPLE 7 Creation of precursor mutant

[0066] To elucidate HS^(act) biosynthesis, mutants defective in theformation of HS^(act) precursors were created. Chinese hamster ovary(CHO) cells were selected as the target cell because wild-type CHO cellsproduce HS^(inact) but not HS^(act) (presumably, due to lack of HS3-O-Sulfotransferase-1 (3-OST-1) expression). Furthermore, a series ofHS biosynthetic mutants have been successfully made in CHO cells(23-28).

[0067] The 3-OST-1 gene, which was presumed to be the terminal enzyme inthe HS^(act) biosynthetic pathway, was introduced into CHO cells byretroviral transduction (29). 3-OST-1 expression gave rise to CHO cellswith the ability to produce HS^(act). A CHO cell line containing 3copies of 3-OST-1 (referred to herein as “3-OST-1 expressing CHO cells,“3-OST-1 triple mutant” or “multi-expresser”) was selected for furtheranalysis and experimentation. The 3-OST-1 triple mutant was subjected tochemical mutagenesis. Cells positive for the desired knockout phenotype,specifically, positive for HS expression (selected by FGF-2 binding) andnegative for HS^(act) expression (selected by AT binding), wereidentified and isolated (FIG. 5, panels E and F). This cell line isreferred to herein as the “6-OST-1 deficient mutant”or “precursormutant.”

[0068] The precursor mutant disclosed herein, which makes decreasedamounts of 6-O-sulfated residues, is defective in AT binding (FIG. SE)due to decreased 6-O-sulfotransferase activities. The defect in thismutant has been corrected, both in vivo (by transduction with a 6-OST-1gene) and in vitro (by contacting HS with 6-OST-1 protein).

EXAMPLE 8 Correction of the precursor mutant

[0069] The 6-OST-1 sulfate defect of the 6-OST-1 deficient mutant wascorrected (i.e., the phenotype of the parental cell line was recovered)by transduction with ⁶-O-sulfotransferase-1 gene (FIG. 5, panels G andH). The resultant cell line (the “Correctant”) produced HS, 50% of whichis HS^(act). Previously reported cell lines have been observed toproduce less than 1% ^(HSact.) This represents the highest percentage ofHS^(act) production by any reported cell line. Thus, the presentinvention provides for a cell line that produces high yields ofHS^(act), as well as methods of efficiently producing HS^(act). Thiscell line (termed “hyper-producer”) expresses approximately 28% -50% ofHS^(act) relative to HS^(total.)

EXAMPLE 9 GAGs from precursor defective mutant and wild-type CHO cellshave similar charge densities.

[0070] GAGs from the 3-OST-1 expressing CHO cells and precursor mutantcells were isolated and analyzed by biosynthetic labeling studies using[6-³H]GlcN. HPLC anion-exchange analysis of the [³H]GAG chains from theprecursor mutant resolved HS (0.31-0.50 M NaCl) from chondroitin sulfate(0.52-0.60 M NaCl) (FIG. 6). The GAG chains from the 3-OST-1 expressingCHO cells (FIG. 6, solid tracer) resolved into a similar profile to thatof the precursor mutant (FIG. 6, broken tracer). This result impliesthat the HS from the precursor mutant and the 3-OST-1 expressing CHOcells have similar charge densities charge density and therefore, thedecrease in AT-binding activity observed in the precursor mutant may beattributed to structural changes in the HS, possibly due to differencesin degree of sulfation.

EXAMPLE 10 Precursor mutant makes less 6-O-sulfate containingdisaccharides than 3-OST-1 expressing CHO cells.

[0071] Since HS from the precursor mutant has similar charge density tothat of the 3-OST-1 expressing CHO cells, the decrease in AT binding inthe precursor mutant was expected to correlate with a change in thestructure of the HS chains. The GAGs synthesized by the 3-OST-1expressing CHO cells and precursor mutant were analyzed by biosyntheticlabeling studies using [³⁵S]sulfate. The 3-OST-1 expressing CHO cellsand precursor mutant cells produced the same amount of [³⁵S]HS and bothsamples contained ˜70% HS and ˜30% chondroitin sulfate (data not shown).This ratio of HS to chondroitin sulfate is consistent with the resultshown in FIG. 6 (wherein HS accounted for 68% of the GAGs in theprecursor mutant, and HS accounted for 66% of the GAGs in the 3-OST-1expressing CHO cells). [³⁵S]sulfate labeled HS chains from the 3-OST-1expressing CHO cells and precursor mutant cells were then digested witha mixture of heparitinases. The resulting disaccharides (representingapproximately 93% of total [³⁵S]sulfate counts) were separated on aBio—Gel P2 column and further resolved by IPRP-HPLC with appropriateinternal standards. As Table 5 shows, the precursor mutant cellsproduced reduced amounts of 6-O-sulfated disaccharides relative to the3-OST-1 expressing CHO cells. TABLE 5 Disaccharide Composition 3-OST-1expressing Disaccharide CHO cells Precursor Mutant Correctant ΔUA-GlcNS30% 35% 27% ΔUA-GlcNAc6S  7%  5%  9% ΔUA-GlcNS6S  9%  4% 13% ΔUA2S-GlcNS20% 36% 22% ΔUA2S-GlcNS6S 34% 19% 29%

EXAMPLE 11 Precursor mutant and 3-OST-expressing CHO cells express6-OST-1 mRNA, but not 6-OST-2 mRNA or 6-OST-3 mRNA.

[0072] In order to explain the reduced levels of 6-O-sulfate containingdisaccharides in the precursor mutant, 6-OST-1 isoform expression in3-OST-1 expressing CHO cells was compared with 6-OST-1 isoformexpression in the precursor mutant cells. Human 6-OST-1, 6-OST-2, and6-OST-3 cDNA were used as probes in Northern blot and RT-PCR studies.Northern analysis indicated that the precursor mutant and the 3-OST-1expressing CHO cells have the same level of 6-OST-1 mRNA. However, no6-OST-2 or 6-OST-3 mRNA was detected in either the 3-OST-1 expressingCHO cells or the precursor mutant cells, indicating that CHO cellsexpress 6-OST-1 only.

[0073] The expression pattern for 6-OST isoforms in CHO cells wasconfirmed using RT-PCR analysis of 3-OST-1 expressing CHO cells andprecursor mutant cells. One set of PCR primers for 6-OST-1, three setsof PCR primers for 6-OST-2, and two sets of PCR primers for 6-OST-3 wereused to evaluate mRNA expression of the 6-OST isoforms. The same levelof 6-OST RT-PCR products was observed for both 3-OST-1 expressing CHOcells and the precursor mutant cells; however, no RT-PCR products wereobserved in either cell from the three sets of 6-OST-2 RT-PCR reactionsand two sets of 6-OST-3 RT-PCR reactions. The Northern blot and RT-PCRanalyses described above demonstrate that CHO cells express 6-OST-1, butnot 6-OST-2 or 6-OST-3.

EXAMPLE 12 The coding region of 6-OST-1 in the precursor mutant containsno point mutations.

[0074] Northern blot and RT-PCR analysis indicated that the precursormutant cells and the 3-OST-1 expressing CHO cells express similar levelsof 6-OST-1 mRNA, however, as described in greater detail below, thelevel of 6-OST-1 activity in the precursor mutant is lower than thelevel of activity in the 3-OST-1 expressing CHO cells. This observationraised the possibility that the precursor mutant CHO cells might haveone or more point mutation(s) in 6-OST-1 gene that diminishes the levelof 6-OST sulfotransferase activity. The coding regions of 6-OST-1 RT-PCRproducts from the mutant were double-strand-sequenced and no pointmutation was observed compared to wild-type 6-OST-1. Thus, thediminished level of 6-OST activity observed is not due to a defect inthe 6-OST-1 gene in the precursor mutant.

EXAMPLE 13 Precursor mutant has decreased 6-O-sulfotransferaseactivities compared to wild-type CHO cells.

[0075] FACS analysis showed that the precursor mutant cells weredefective in AT binding (FIG. 5, panel E). The coding sequence of6-OST-1 was not mutated and 6-OST-1 mRNA expression levels were normal;however, disaccharide compositional studies demonstrated that theprecursor mutant made less 6-O-sulfated residues in vivo than wild-typeCHO cells.

[0076] The 6-O-sulfotransferase activity of the precursor mutant wasevaluated in vitro. Crude cell homogenates from wild-type CHO cells andprecursor mutant CHO cells served as the source of 6-OST-1 enzyme. HSderived from wild-type CHO cells, N,O-desulfated, re-N-sulfated heparin(CDSNS-heparin), and 6-O-desulfated heparin was incubated with 6-OST-1enzyme from wild-type CHO cells and precursor mutant in the presence ofa sulfate donor. The resulting reaction products were digested by acombination of heparitinases, followed by Bio Gel P2 chromatography. Thedisaccharides collected were then subjected to IPRP-HPLC analysis. Both2-O-[³⁵S] sulfate (control) and 6-O-[³⁵S]sulfate labeled disaccharidesresulting from the 6-OST-1 enzymes were quantitated.2-O-sulfotransferase activity was similar in precursor mutant cells(118±3 pmol/min/mg) and the wild-type CHO cells (122±2 pmol/min/mg) whenCDSNS-heparin was used as substrate (not shown). However, a 30% to 39%reduction of 6-O-sulfotransferase activity was observed in the precursormutant relative to the wild-type CHO cells with all three substrates(Table 6). TABLE 6 6-O-sulfotransferase activity (pmol/min/mg) %reduction of wild-type activity Wild-type Precursor in precursorSubstrate CHO Mutant mutant HS (CHO K1) 5.6 ± 0.3 3.9 ± 0.4 30%6-O-desulfated heparin 4.4 ± 0.3 2.7 ± 0.5 39% CDSNS-heparin 11 ± 2  7 ±1 38%

EXAMPLE 14 Size exclusion chromatography coupled with mass spectrometryis effective for compositional analysis of oligosaccharides.

[0077] Mass spectrometric detectors produce far more information thanconventional UV or fluorescent detectors and allows the monosaccharidecomposition of individual components to be determined (39). Introducingstable isotope PAP³⁴S into the 3-O-position of HS by pure 3-OST-1, a3-O-sulfate containing disaccharide with a unique mass was identifiedusing a combination of capillary IPRP-HPLC coupled with massspectrometry. The method consumes 0.5 μg of total HS for separating anddetecting different HS disaccharides. This method provides a practicalway of accomplishing HS disaccharide analysis of general HS samples fromcells or tissues without radioisotope labeling. Furthermore,biologically inactive HS oligosaccharides could be treated in vitro withdifferent pure sulfotransferases plus stable sulfur isotope PAPS (e.g.,PAP³³S and PAP³⁴S). The different stable isotope tagged biologicallyactive oligosaccharides could then be sequenced by a combination ofcapillary IPRP-HPLC for separation and mass spectrometry. In thismanner, biologically critical regions can be pinpointed and sequenced.

[0078] Capillary IPRP-HPLC coupled with mass spectrometry. Heparinmolecules exhibiting a high affinity for a synthetic peptide(CRPKAKAKAKAKDQTK) mimicking a heparin-binding domain of heparininteracting protein (HIP) also show an extremely high affinity for AT(37). It was expected that inclusion of this small peptide in theheparitinase digestion solution would protect 3-O-[C5S]sulfate labeledHS from degrading into tetrasaccharides. Theoretically, HIPpeptide-protected, AT binding HS oligosaccharides would be recovered.However, in the presence of the HIP peptide, all the 3-O-[³⁵S]sulfatelabeled sugars were degraded into disaccharides instead ofoligosaccharides or tetrasaccharides as judged by their elution positionon Bio—Gel P2 and their unique elution positions on IPRP-HPLC (the major3-O-[³⁵S]sulfate containing disaccharides eluted right beforeΔUA—GlcNS6S disaccharide standard). Because there is no ΔUA—GlcNS3Sstandard reported, the structure was verified. Stable isotope PAP³⁴S wasmade. The PAP³⁴S (99% isotope purity determined by ES-MS) was preparedby incubating ATP and stable isotope Na₂ ³⁴SO₄ (Isonics Corp.) with ATPsulfurylase (Sigma), adenosine 5′-phosphosulfate kinase (a generous giftfrom Dr. Irwin H. Segel), and inorganic pyrophosphatase (Sigma) (38 ).HS chains from wild-type CHO cells were labeled with pure 3-OST-1 plusPAP³⁴S. A capillary IPRP-HPLC (LC Packings) method for separating HSdisaccharides was developed. This method is similar to conventionalIPRP-HPLC (29) except using 5 mM dibutylamine as an ion pairing reagent(Sigma), and then coupled it to an ESI-TOF-MS (Mariner Workstation,PerSeptive Biosystems, Inc.) to detect the mass of each disaccharideeluted. Six HS disaccharide standards from Seikagaku were separated bycapillary HPLC and detected by negative polarity ESI-MS. The accuracy ofthe ES-MS is ±0.001 m/z unit after calibration with the molecularstandard sets supplied by the manufacture (Bis TBA,Heptadecaflurononanoic acid, Perflurotetradecanoic acid).3-O-³⁴S-labeled HS was digested with a combination of 1 mU of eachheparitinase I, heparitinase II, heparitinase IV, and heparinase in theabsence or presence of 0.5 mg/ml HIP peptide. 0.5 μg of digested HS wasinjected into capillary HPLC coupled with mass spectrometry (FIG. 8). UVpeak B eluted at the same time as a ΔUA—GlcNS6S standard, whereas UVpeak D eluted at the same time as a ΔUA2S—GlcNS standard (FIG. 8, panelA). Three major ions with m/z 247.5, 496.0, and 625.2 were observed inboth UV peaks (FIG. 3, panel B and D), where 496.0 is z1 (−1) charged,247.5 is z2 (−2) charged, and 625.2 is one dibutylamine adducted, z1(−1) charged ΔUA—GlcNS6S or ΔUA2S—GlcNS disaccharides. However, when m/zregions 494.0 to 501.0 from both peal B and peak D were expended (panelC and panel E), a non-natural abundant, z1 charged molecular ion withm/z 498.0 was observed in UV peal B, but not in UV peak D. 498.0 vs.496.0 of disaccharide ions should represent ΔUA—GlcNS3[³⁴5]S andΔUA—GlcNS6S, respectively. The mass for ΔUA—GlcNS3[³⁴]S is barelydetectable in the absence of HIP peptide, which is consistent with theliterature that 3-O-sulfate containing sugars are usually degraded intotetrasaccharides not disaccharides by a mixture of heparitinasedigestion (20,33). HIP peptide was included in heparitinase digestionwhen 3-O-containing HS were degraded into disaccharides.

[0079] Materials and Methods for Practicing the Inventions ExemplifiedAbove

[0080] Cell Culture. Wild-type Chinese hamster ovary cells (CHO-K1) wereobtained from the American Type Culture Collection (CCL-61; ATCC,Rockville, Md.). CHO cells were maintained in Ham's F-12 mediumsupplemented with 10% fetal bovine serum (HyClone), penicillin G (100units/ml), and streptomycin sulfate (100 μg/ml) at 37 C under anatmosphere of 5% CO₂ in air and 100% relative humidity. The cells werepassaged every 34 days with 0.125% (w/v) trypsin and 1 mM EDTA, andafter 10-15 cycles, fresh cells were revived from stocks stored underliquid nitrogen. Low-sulfate medium was composed of Ham's F-12 mediumsupplemented with penicillin G (100 units/ml) and 10% fetal bovine serumthat had been dialyzed 200-fold against phosphate-buffered saline (30).Low-glucose Ham's F-12 medium contained 1 mM glucose supplemented withpenicillin G (100 units/ml), streptomycin sulfate (100 μg/ml), and fetalbovine serum that had been dialyzed 200-fold against phosphate-bufferedsaline (30). All tissue culture media and reagents were purchased fromLife Technologies (Gaithersburg, Md.) unless otherwise indicated.

[0081] 3-OST-1 recombinant retroviral transduction. The retrovirusplasmid pMSCVpac was obtained from Dr. Robert Hawley, University ofToronto (31). pCMV3-OST-1 was digested with BglII and XhoI to releasethe wild-type murine 3-OST-1 cDNA (15). The cDNA fragment (1,623 bp) wascloned into the BglII+XhoI sites in pMSCVpac. All plasmid DNA preparedfor transfection was made with the Invitrogen SNAP-MIDI kit according tothe manufacturer's directions. Infectious virions were produced bytransducing ecotropic PHOENIX packaging cells with recombinant provirusplasmids using the calcium phosphate transfection technique. Followingthe precipitation step, the cells were re-fed with 2 ml/well of fleshDMEM and incubated overnight. Viral supernatants were collected, eitherflash-frozen in liquid nitrogen, and stored at −80° C. or used directlyafter low-speed centrifugation.

[0082] Wild-type CHO cells containing ecotropic receptors were treatedwith trypsin and then plated at 150,000 cells/well in a 6-well dish. Oneday later, target cells (<70% confluent) were incubated overnight withviral supernatants containing 5 μg/ml Polybrene surfactant. After 12hours, the virus containing media was replaced with fresh growth media.Wild-type CHO cells were exposed to recombinant retrovirus three timesand selected and maintained in 7.5 μg/ml puromycin (Sigma).

[0083] Antithrombin and FGF-2 labeling. The standard reaction mixturefor preparing fluorescent AT contained 20 mM NaH₂PO₄ (pH 7.0), 0.3 mMCaCl₂, 25 μg of PBS dialyzed AT (GlycoMed), 4 mU neuraminidase(Worthington Biochemical Corp.), 4 mU galactose oxidase (WorthingtonBiochemical Corp.), and 125 μg/ml fluorescein hydrazide (MolecularProbe, C-356) in a final volume of 280 μl. The mixtures were incubatedat 37° C. for 1 h. PBS (1 ml) and a 50% slurry of heparin-Sepharose inPBS (100 μl) was added and mixed end-over-end for 20 min. Aftercentrifugation, the heparin-Sepharose beads were washed 4 times with PBS(1 ml). Labeled AT was eluted with four 0.25 ml aliquots of 10×concentrated PBS and desalted by centrifugation for 35 minutes at 14,000rpm through two Microcon-10 columns (Millipore). The concentrated AT wasdiluted with 0.5 ml 10% FBS in PBS containing 2 mM EDTA and useddirectly for cell labeling studies.

[0084] Fluorescent FGF-2 was prepared by mixing 50 μl of 1 M sodiumbicarbonate to 0.5 ml of PBS containing 2 mg/ml BSA and 3 μg FGF-2. Themixture was then transferred to a vial of reactive dye (Alexa 594,Molecular Probes) and stirred at room temperature for 1 hour. Theisolation of the labeled FGF-2 was identical to that described above forlabeled AT.

[0085] Cell sorting. Nearly confluent monolayers of 3-OST-1 transducedCHO K1 cells were detached by adding 10 ml of 2 mM EDTA in PBScontaining 10% FBS and centrifuged. The cell pellets were placed on iceand 50 μl each of fluorescein-AT and Alexa 594-FGF-2 were added. After30 minutes, the cells were washed once and resuspended in 1 ml of 10%FBS in PBS containing 2 mM EDTA. Flow cytometry and cell sorting wasperformed on FACScan and FACStar instruments (Becton Dickinson) usingdual color detection filters. AT and FGF-2 binding positive cells weresorted and subsequently single-cell cloned into a 96 well plate. Thesingle cell clones were expanded and frozen for further analysis.

[0086] Twelve 3-OST-1 transduced CHO K1 clones were obtained asdescribed above. The number of copies of 3-OST-1 in the individualclones was determined by Southern analysis as follows. Genomic DNA (10jig) was digested with 40 U of EcoRI overnight at 37° C.,electrophoresed on a 0.7% (w/v) agarose gel, transferred to GeneScreenPlus (NEN) and probed with 3-OST-1 cDNA labeled with the Megaprimelabeling kit (Amersham). Blots were hybridized in ExpressHyb Solution(Clontech) containing 3-OST-1 probe (2×10⁶ cpm/ml), followed byautoradiography. The cell clone with 3 copies of 3-OST-1 was expandedand frozen for further studies.

[0087] Mutant screening. Wild-type CHO with 3 copies of 3-OST-1 weremutagenized with ethylmethane sulfonate as described in the literature(31) and frozen under liquid nitrogen. A portion of cells was thawed,propagated for 3 days, and labeled with both Alexa 594-FGF-2 andfluorescein-AT. The labeled cells were sorted and FGF-2 positive and ATnegative cells were collected. Approximately 1×10⁴ sorted cells werecollected into 1 ml of complete F-12 Ham's media, then plated in T-75flasks. Sorted cell populations were maintained in complete F-12 Ham'smedium for one week, then the cells were labeled and sorted again asdescribed above. After 5 rounds of sorting, FGF-2 positive and ATnegative cells were single-cell-sorted into a 96 well plate. The singlecell clones were expanded and frozen for further analysis. The sortingprofiles of CHO K1 with 3 copies of 3-OST-1, precursor mutant, and the6-OST-1 correctant of the mutant were shown by dual-color fluorescenceflow cytometric analysis in FIG. 5.

[0088] HS Preparation and analysis. Cell monolayers were labeledovernight with 100 μCi/ml of carrier free sodium [³⁵S]sulfate (ICN) insulfate deficient DMEM, supplemented with penicillin G (100 Units/ml),and 10% (v/v) dialyzed FBS. The proteoglycan fraction was isolated byDEAE-Sepharose chromatography and beta-eliminated in 0.5 M NaBH₄ in 0.4M NaOH at 4° C. overnight. The samples were neutralized with 5 M aceticacid until bubble formation ceased and the released chains were purifiedby another round of DEAE-Sepharose chromatography followed by ethanolprecipitation. The pellet from centrifugation was washed with 75%ethanol and resuspended in water. The GAGs were digested with 20 mU ofchondroitinase ABC (Seikagaku, Inc.) in buffer containing 50 mM Tris—HCland 50 mM sodium acetate (pH 8.0). Complete digestion of chondroitinsulfate by chondroitinase ABC was assured by monitoring the extent ofconversion of the carrier to disaccharides (100 μg=1.14 absorbance unitsat 232 nm). HS was purified from chondroitinase degraded products byphenol/chloroform (1:1, v/v) extraction and ethanol precipitation. Afterwashing the pellets with 0.5 ml of 75% ethanol, the HS was dissolved inwater for further analysis.

[0089] cDNA cloning and expression of CHO 6-OST-1. Sequences coding forCHO 6-OST-1 were amplified from a CHO K1/cDNA quick-clone library(Clontech). The reaction mixture contained 2 units pfupolymerase(Stratagene), 1 ng of cDNA, and 100 pmol of the Primers. Thesense primer has an added Bgl II site (5′ GCAGATCTGCAGGACCATGGTTGAGCGCGCCA GCAAGTTC-3′) and the antisense primer has an added Xba I site(5′-GCTCTAGACTACCACT TCTCAATGATGTGGCTC-3′). The 6-OST-1 primer sequencesare derived from the human 6-OST-1 cDNA sequence (from residue 240 to264) and to the complement of this sequence ( from residue 1147 to 1172)as reported (32). After 30 thermal cycles (1 min of denaturation at 94°C. 2 min of annealing at 55° C., 3 min of extension at 72° C.), theamplification products were analyzed in 1% agarose gels and detected byethidium bromide staining. The amplification products were excised fromthe gel and cleaned by Gel Extaction kit (Qiagen). The PCR product wastreated with Bgl II and Xba I, ligated into Xba I and BamH1 digestedpInd/Hygro plasmid (Clontech) and transformed into E.coli DH5α competentcells. Four clones from each of two separate PCR reactions weresequenced and found to be identical. pInd/Hygro 6-OST-1 containingplasmid was transfected into the CHO mutant cells. AT and FGF-2 bindingpositive cells were sorted and subsequently single-cell-cloned into a 96well plate. The single cell clones were expanded and frozen for furtheranalysis.

[0090] 6-O-sulfation of HS in vitro. The standard reaction mixturecontained 50 mM MES (pH 7.0), 1% (w/v) Triton X-100, 5 mM MnCl₂, 5 mMMgCl₂, 2.5 mM CaCl₂, 0.075 mg/ml protamine chloride, 1.5 mg/ml BSA,either metabolically labeled [³⁵S]HS or non-radioactive HS chains, coldPAPS (0.5 mM) or [³⁵S]PAPS (25 μM, 2×10⁷ cpm), and 70 ng of purifiedbaculovirus-expressed human 6-OST-1 in a final volume of 50 μl. Themixtures were incubated either 20 minutes or overnight at 37° C., and200 μg of chondroitin sulfate C was added. HS chains were purified byphenol/chloroform extraction and anion exchange chromatography on0.25-ml columns of DEAE-Sephacel packed in 1 ml syringes (20). Afterethanol precipitation, the pellets were washed with 75% ethanol, driedbriefly under vacuum, and dissolved in water for further analysis.

[0091] Separation of HS^(act) and HS^(inact) by AT-affinitychromatography. AT-HS complexes were created by mixing 3-O-sulfated HSin 500 μl of HB buffer (150 mM NaCl, 10 mM Tris—Cl (pH 7.4)) with 2.5 mMAT, 100 μg of chondroitin sulfate, 0.002% Triton-X 100, and 1 mM each ofCaCl₂, MgCl₂, and MnCl₂ (18). HB containing ˜50% slurry of ConcanavalinA-Sepharose 4B (60 μl) was then added. AT complexes were bound toConcanavalin A by way of the Asn-linked oligosaccharides. After one hourend-over-end rotation at 4° C., the beads were sedimented bycentrifugation at 10,000×g. The supernatant was collected and the beadswere washed three times with 1.25 ml of HB containing 0.0004% Triton-X100. The supernatant and washing solutions contained HS^(inact). TheHS^(act) was eluted with three successive washes with 100 μl HBcontaining 1 M NaCl and 0.0004% Triton-X 100. After adding 100 μg ofchondroitin sulfate as carrier to HS^(act), the sample was extractedwith an equal volume of phenol/chloroform, followed by chromatography onDEAE-Sepharose and ethanol precipitation. The pellets were washed with75% ethanol, dried briefly under vacuum and dissolved in water.

[0092] Disaccharide analysis of HS. Heparitinase I (EC. 4.2.2.8),heparitinase II (no EC number), and heparinase (EC. 4.2.2.7) wereobtained from Seikagaku heparitinase IV was obtained from Dr. Yoshida,Seikagaku Corporation, Tokyo. Heparitinase I recognizes the sequences:GlcNAc/NS±6S(3S?)—↓GlcUA—GlcNAc/NS±6S. The arrow indicates the cleavagesite. Heparitinase II has broad sequence recognition:GlcNAc/NS±6S(3S?)—↓GlcUA/IdoUA±2S—GlcNAc/NS±6S. Heparinase(heparitinaseIII) and heparitinase IV recognize the sequences:GlcNS±3S±6S—↓IdoUA2S/GlcUA2S—GlcNS±6S. The reaction products andreferences can be found in the following references (33,34). Thedigestion of HS^(act) was carried out in 100 μl of 40 mM ammoniumacetate (pH 7.0) containing 3.3 mM CaCl₂ with 1 mU of heparitinase I or1 mU of each heparitinase I, heparitinase II, heparitinase IV, andheparinase (heparitinase III). The digestion was incubated at 37° C.overnight unless otherwise indicated. For low pH nitrous aciddegradation, radiolabeled HS samples were mixed with 10 ,g bovine kidneyHS (ICN) and digested (35).

[0093] Disaccharides were purified by Bio—Gel P2 chromatography andresolved by ion pairing reverse-phase HPLC with appropriate disaccharidestandards (36). Bio—Gel P2 or P6 columns (0.75×200 cm) were equilibratedwith 100 mM ammonium bicarbonate. Radiolabeled samples (200 μl) weremixed with Dexdran blue (5 μg) and phenol red (5 μg) and loaded on thecolumn. The samples were eluted at a flow rate of 4 ml/hour withcollection of 0.5 ml fractions. The desired fractions were dried undervacuum, individually or pooled to remove ammonium bicarbonate.

[0094] Capillary IPRP-HPLC coupled with mass spectrometry. Heparinmolecules exhibiting a high affinity for a synthetic peptide(CRPKAKAKAKAKDQTK) mimicking a heparin-binding domain of heparininteracting protein (HIP) also show an extremely high affinity for AT(37). It was expected that inclusion of this small peptide in theheparitinase digestion solution would protect 3-O-[³⁵S]sulfate labeledHS from degrading into tetrasaccharides. Theoretically, HIPpeptide-protected, AT binding HS oligosaccharides would be recovered.However, in the presence of the HIP peptide, all the 3-O-[³⁵S]sulfatelabeled sugars were degraded into disaccharides instead ofoligosaccharides or tetrasaccharides as judged by their elution positionon Bio—Gel P2 and their unique elation positions on IPRP-HPLC (the major3-O-[³⁵S]sulfate containing disaccharides eluted right beforeΔUA—GlcNS6S disaccharide standard). Because there is no ΔUA—GlcNS3Sstandard reported, the structure was verified. Stable isotope PAP³⁴S wasmade. The PAP³⁴S (99% isotope purity determined by ES-MS) was preparedby incubating ATP and stable isotope Na₂ ³⁴SO₄ (Isonics Corp.) with ATPsulfurylase (Sigma), adenosine 5′-phosphosulfate kinase (a generous giftfrom Dr. Irwin H. Segel), and inorganic pyrophosphatase (Sigma) (38). HSchains from wild-type CHO cells were labeled with pure 3-OST-1 plusPAP³⁴S. A capillary IPRP-HPLC (LC Packings) method for separating HSdisaccharides was developed. This method is similar to conventionalIPRP-HPLC (29) except using 5 mM dibutylamine as an ion pairing reagent(Sigma), and then coupled it to an ESI-TOF-MS (Mariner Workstation,PerSeptive Biosystems, Inc.) to detect the mass of each disaccharideeluted. Six HS disaccharide standards from Seikagaku were separated bycapillary HPLC and detected by negative polarity ESI-MS. The accuracy ofthe ES-MS is ±0.001 m/z unit after calibration with the molecularstandard sets supplied by the manufacture (Bis TBA,Heptadecaflurononanoic acid, Perflurotetradecanoic acid).3-O-³⁴S-labeled HS was digested with a combination of 1 mU of eachheparitinase I, heparitinase II, heparitinase IV, and heparinase in theabsence or presence of 0.5 mg/ml HIP peptide. 0.5 μg of digested HS wasinjected into capillary HPLC coupled with mass spectrometry (FIG. 8). UVpeal B eluted at the same time as a ΔUA—GlcNS6S standard, whereas UVpeak D eluted at the same time as a ΔUA2S—GlcNS standard (FIG. 8, panelA). Three major ions with m/z 247.5, 496.0, and 625.2 were observed inboth UV peaks (FIG. 3, panel B and D), where 496.0 is z1 (−1) charged,247.5 is z2 (−2) charged, and 625.2 is one dibutylamine adducted, z1(−1) charged ΔUA—GlcNS6S or ΔUA2S—GlcNS disaccharides. However, when m/zregions 494.0 to 501.0 from both peak B and peak D were expended (panelC and panel E), a non-natural abundant, z1 charged molecular ion withm/z 498.0 was observed in UV peak B, but not in UV peak D. 498.0 vs.496.0 of disaccharide ions should represent ΔUA—GlcNS3[³⁴5]S andΔUA—GlcNS6S, respectively. The mass for ΔUA—GlcNS3[³⁴S]S is barelydetectable in the absence of HIP peptide, which is consistent with theliterature that 3-O-sulfate containing sugars are usually degraded intotetrasaccharides not disaccharides by a mixture of heparitinasedigestion (20,33). HIP peptide was included in heparitinase digestionwhen 3-O-containing HS were degraded into disaccharides.

[0095] Northern blot hybridization and RT-PCR. To generate specificNorthern blot hybridization probes, PCR primers were designed thatbracket unique sequences within human 6-OST-1, 6-OST-2 and 6-OST-3. A249 bp PCR product that corresponds to a region within the 3′-UTR of the60ST-1 gene starting at position 1772 and ending at 2021 was used as anisoform specific probe. Similarly, a 299 bp PCR product that correspondsto a region in the 3′-UTR of the 60ST-2 gene starting at position 1831and ending at 2130, and another product within the 3′-UTR of the 6-OST-3gene starting at 943 and ending at 1378 (444 bp) were used as a probe.PCR was performed with α[32P] dCTP (NEN Life Science Products) andisoform-specific radio-labeled probes were purified on G-25 Sephadexspin columns (Boehringer Mannheim). Hybridizations were carried out asto the manufacturer's instructions using 2×10⁶ cpm probe per ml ofExpressHyb solution (CLONTECH). After the hybridizations were complete,the blots were washed twice in 2×SSC containing 0.1% SDS and once with0.1×SSC containing 0.1% SDS, all at room temperature. Blots were thenwashed with 0.1X SSC containing 0.1% SDS at 50° C. For blots hybridizedwith the 6-OST probe, this last wash was repeated twice at 65° C. Themembranes were then subjected to autoradiography with BioMax imagingfilm (Kodak) with a BioMax MS intensifying screen (Kodak).

[0096] For RT-PCR, poly A purified or DNase I treated total RNA wasused. Primer pairs were designed that bracket isoform specific regionswithin the human sequences for both 60ST-2, and 60ST-3. For 60ST-1, a569 bp fragment corresponding to nt 54 (GCG TGC TTC ATG CTC ATC CT) to622 (GTG CGC CCA TCA CAC ATG T) within the hamster sequence was used.For 60ST-2, PCR targets included regions starting at nt 23 (CTG CTG CTGGCT TTG GTG AT) and 346 (GCA GAA GAA ATG CAC TTG CCA) and ending at nt1471 (GCC GCT ATC ACC TTGTCC CT), 1491 (TCA TTG GTG CCA TTG CTG G) and1532 (TGA GTG CCA GTT AGC GCC A). For 60ST-3, the targets includedregions that start at nt 5 (CCG GTG CTC ACT TTC CTC TTC) and 353 (TTCACC CTC AAG GAC CTG ACC) and end at nt 988 (GCT CTG CAG CAG GAT GGT GT)and 1217 (GCT GGA AGA GAT CCT TCG CAT AC). Total RNA was purified fromwild-type and precursor mutant CHO-K1 cells using the RNeasy total RNAkit from Qiagen as to the manufacturer's instructions. RNA wasquantitated by absorbance at 260 nm and 100 μg of total RNA was reactedwith DNase I (Ambion) at 37° C. for 45 minutes, twice extracted withequal volumes of acid phenol/chloroform, precipitated in ethanol, andreconstituted in DEPC treated water. Further selection of poly-A plusRNA was carried out with the Oligotex mRNA kit (Qiagen). RNA integritywas checked after electrophoresis on a 1% agarose gel and all RTreactions were run with M-MLV reverse transcriptase (Ambion) accordingto manufacturer's instructions. PCR was performed with Super Taqpolymerase (Ambion).

[0097] Baculovirus expression and Purification of 6-OST-1. Human 6-OST-1recombinant baculovirus was prepared using the pFastBas HT donor plasmidmodified by the insertion of honeybee mellitin signal peptide (36) andthe Bac-to-Bac Baculovirus expression system (Life Technologies, Inc.)according to the manufacturer's protocol, except that recombinant bacmidDNA was purified using an endotoxin-free plasmid purification kit(Qiagen, Inc.) and transfection of Sf9 cells was scaled up to employ 3μg of bacmid DNA and 6×10⁶ exponentially-growing cells in a 100-mm dish.At day three post-transfection, baculovirus was precipitated from themedium with 10% PEG, 0.5 M NaCl at 12,000×g, re-suspended in 14 ml ofmedium, and applied to a 100-mm dish seeded with 1.5×10⁷ Sf9 cells.Medium from the infected cells was harvested after 90 hours of growth at27° C., centrifuged at 400×g, made to 10 M in Tris, adjusted to pH 8.0,and centrifuged at 4000×g. Clarified medium was diluted with an equalvolume of cold 10 mM Tris—HCl, pH 8.0, and stirred for 30 minutes with0.6 ml (packed volume) of Toyopearl 650M chromatographic media(TosoHaas). The heparin-sepharose was packed into a column (0.4×4.75cm), washed with 5 ml of TCG 50 (10 mM Tris—HCl, pH 8.0, 2% glycerol,0.6% CHAPS, 50 mM NaCl), eluted with 1.2 ml of TCG 1000 (as above, but 1M in NaCl) containing 10 mM imidazole, and concentrated to 0.25 ml in aMicrocon YM-10 centrifugal filter (Millipore Corp.).

[0098] Histidine-tagged recombinant 6-OST-1 was affinity purified bymixing the product eluted from heparin-sepharose for 90 minutes at 4° C.with NiNTA magnetic agarose beads (Qiagen, Inc.) and magneticallysedimented from 60 μl of suspension. The beads were washed twice with0.125 ml of TCG 400 containing 20 mM imidazole and eluted twice with0.03 ml of TCG 400 containing 250 mM imidazole. The combined elutionfractions contained approximately 25% of the sulfotransferase activitypresent in the starting medium.

[0099] Bacterial expression and Unification of 6-OST-1. Expressionvector pET15b was purchased from Novagen (Madison, Wis.). E. colistrains BL21 and DH5a were obtained through ATCC (Manassas, Va.). An AseI restriction site was introduced at 211-216 bp and a BamHI restrictionsite was introduced at 1344-1349 bp of human 6-OST-1-1(32) by PCR. The6-OST-1 gene was then ligated into Nde I and BamHI digested pET15b andtransformed into competent E. coli strain DH5α. A BL2I colony containing6-OST-1 in pET15b with confirmed sequence was used to inoculate 2 L ofLB containing 100 μg/mL ampicillin. The cultures were shaken in flasksat 250 rpm at 37° C. When the optical density at 600 nm reached 1.2, 1mM IPTG was added to the cultures. The cultures were then agitated at250 rpm overnight at room temperature. The cells were pelleted at 5,000rpm for 15 minutes. The supernatant was discarded and the cell pelletwas resuspended in 40 mL of 20 mM Tris, 500 mM NaCl, 0.6% CHAPS, 1%glycerol, and 5 mM imidazole, pH 7.9 (“binding buffer”). The cells werehomogenized, and the homogenate was centrifuged at 13,000 rpm for twentyminutes. The supernatant was filtered through 0.2 μm filter paper andloaded onto a BioCAD HPLC system (PerSeptive Biosystems, Cambridge,Mass.) and purified using Ni²⁺ chelate chromatography. Briefly, thesupernatant was loaded onto the column and washed with binding bufferuntil unbound material was washed off the column. Then, low affinitymaterial was washed off the column using 20 mM Tris, 500 mM NaCl, 0.6%CHAPS, 1% glycerol, and 55 mM imidazole, pH 7.9 and 6-OST-1 was elutedfrom the column with 20 mM Tris, 500 mM NaCl, 0.6% CHAPS, 1% glycerol,and 500 mM imidazole, pH 7.9. The purity of the recombinant 6-OST-1 wasdetermined using a silver stained protein gel.

[0100] The invention disclosed herein may be embodied in other specificforms without departing from the spirit or essential characteristicsthereof. The foregoing embodiments are therefore to be considered in allrespects illustrative rather than limiting of the disclosed invention.The scope of the invention is thus indicated by the appended claimsrather than by the foregoing description, and all changes which withinthe meaning and range of equivalency of the claims are thereforeintended to be embraced herein.

[0101] The following references are incorporated by reference ill theirentirety.

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1 6 1 1305 DNA Homo sapiens 1 cgcggctcag taattgaagg cctgaaacgcccatgtgcca ctgactagga ggcttccctg 60 ctgcggcact tcatgaccca gcggcgcgcggcccagtgaa gccaccgtgg tgtccagcat 120 ggccgcgctg ctcctgggcg cggtgctgctggtggcccag ccccagctag tgccttcccg 180 ccccgccgag ctaggccagc aggagcttctgcggaaagcg gggaccctcc aggatgacgt 240 ccgcgatggc gtggcccaaa cggctctgccccagcagttg ccgcagacca tcatcatcgg 300 cgtgcgcaag ggcggcacgc gcgcactgctggagatgctc agcctgcacc ccgacgtggc 360 ggccgcggag aacgaggtcc acttcttcgactgggaggag cattacagcc acggcttggg 420 ctggtacctc agccagatgc ccttctcctggccacaccag ctcacagtgg agaagacccc 480 cgcgtatttc acgtcgccca aagtccctgagcgagtctac agcatgaacc cgtccatccg 540 gctgctgctc atcctgcgag acccgtcggagcgcgtgcta tctgactaca cccaagtgtt 600 ctacaaccac atgcagaagc acaagccctacccgtccatc gaggagttcc tggtgcgcga 660 tggcaggctc aatgtggact acaaggccctcaaccgcagc ctctaccacg tgcacatgca 720 gaactggctg cgctttttcc cgctgcgccacatccacatt gtggacggcg accgcctcat 780 cagggacccc ttccctgaga tccaaaaggtcgagaggttc ctaaagctgt cgccgcagat 840 caatgcttcg aacttctact ttaacaaaaccaagggcttt tactgcctgc gggacagcgg 900 ccgggaccgc tgcttacatg agtccaaaggccgggcgcac ccccaagtcg atcccaaact 960 actcaataaa ctgcacgaat attttcatgagccaaataag aagttcttcg agcttgttgg 1020 cagaacattt gactggcact gatttgcaataagctaagct cagaaacttt cctactgtaa 1080 gttctggtgt acatctgagg ggaaaaagaattttaaaaaa gcatttaagg tataatttat 1140 ttgtaaaatc cataaagtac ttctgtacagtattagattc acaattgcca tatatactag 1200 ttatattttt ctacttgtta aatggagggcattttgtatt gtttttcatg gttgttaaca 1260 ttgtgtaata tgtctctata tgaaggaactaaactatttc actga 1305 2 307 PRT Homo sapiens 2 Met Ala Ala Leu Leu LeuGly Ala Val Leu Leu Val Ala Gln Pro Gln 1 5 10 15 Leu Val Pro Ser ArgPro Ala Glu Leu Gly Gln Gln Glu Leu Leu Arg 20 25 30 Lys Ala Gly Thr LeuGln Asp Asp Val Arg Asp Gly Val Ala Pro Asn 35 40 45 Gly Ser Ala Gln GlnLeu Pro Gln Thr Ile Ile Ile Gly Val Arg Lys 50 55 60 Gly Gly Thr Arg AlaLeu Leu Glu Met Leu Ser Leu His Pro Asp Val 65 70 75 80 Ala Ala Ala GluAsn Glu Val His Phe Phe Asp Trp Glu Glu His Tyr 85 90 95 Ser His Gly LeuGly Trp Tyr Leu Ser Gln Met Pro Phe Ser Trp Pro 100 105 110 His Gln LeuThr Val Glu Lys Thr Pro Ala Tyr Phe Thr Ser Pro Lys 115 120 125 Val ProGlu Arg Val Tyr Ser Met Asn Pro Ser Ile Arg Leu Leu Leu 130 135 140 IleLeu Arg Asp Pro Ser Glu Arg Val Leu Ser Asp Tyr Thr Gln Val 145 150 155160 Phe Tyr Asn His Met Gln Lys His Asp Pro Tyr Pro Ser Ile Glu Glu 165170 175 Phe Leu Val Arg Asp Gly Arg Leu Asn Val Asp Tyr Lys Ala Leu Asn180 185 190 Arg Ser Leu Tyr His Val His Met Gln Asn Trp Leu Arg Phe PhePro 195 200 205 Leu Arg His Ile His Ile Val Asp Gly Asp Arg Leu Ile ArgAsp Pro 210 215 220 Phe Pro Glu Ile Gln Lys Val Glu Arg Phe Leu Lys LeuSer Pro Gln 225 230 235 240 Ile Asn Ala Ser Asn Phe Tyr Phe Asn Lys ThrLys Gly Phe Tyr Cys 245 250 255 Leu Arg Asp Ser Gly Arg Asp Arg Cys LeuHis Glu Ser Lys Gly Arg 260 265 270 Ala His Pro Gln Val Asp Pro Lys LeuLeu Asn Lys Leu His Glu Tyr 275 280 285 Phe His Glu Pro Asn Lys Lys PhePhe Glu Leu Val Gly Arg Thr Phe 290 295 300 Asp Trp His 305 3 401 PRTHomo sapiens 3 Met Val Glu Arg Ala Ser Lys Phe Val Leu Val Val Ala GlySer Val 1 5 10 15 Cys Phe Met Leu Ile Leu Tyr Gln Tyr Ala Gly Pro GlyLeu Ser Leu 20 25 30 Gly Ala Pro Gly Gly Arg Ala Pro Pro Asp Asp Leu TyrLeu Phe Pro 35 40 45 Thr Pro Asp Pro His Tyr Glu Lys Lys Tyr Tyr Phe ProVal Arg Glu 50 55 60 Leu Glu Arg Ser Leu Arg Phe Asp Met Lys Gly Asp AspVal Ile Val 65 70 75 80 Phe Leu His Ile Gln Lys Thr Gly Gly Thr Thr PheGly Arg His Leu 85 90 95 Val Gln Asn Val Arg Leu Glu Val Pro Cys Asp CysArg Pro Gly Gln 100 105 110 Lys Lys Cys Thr Cys Tyr Arg Pro Asn Arg ArgGlu Thr Trp Leu Phe 115 120 125 Ser Arg Phe Ser Thr Gly Trp Ser Cys GlyLeu His Ala Asp Trp Thr 130 135 140 Glu Leu Thr Asn Cys Val Pro Gly ValLeu Asp Arg Arg Asp Ser Ala 145 150 155 160 Ala Leu Arg Thr Pro Arg LysPhe Tyr Tyr Ile Thr Leu Leu Arg Asp 165 170 175 Pro Val Ser Arg Tyr LeuSer Glu Trp Arg His Val Gln Arg Gly Ala 180 185 190 Thr Trp Lys Thr SerLeu His Met Cys Asp Gly Arg Thr Pro Thr Pro 195 200 205 Glu Glu Leu ProPro Cys Tyr Glu Gly Thr Asp Trp Ser Gly Cys Thr 210 215 220 Leu Gln GluPhe Met Asp Cys Pro Tyr Asn Leu Ala Asn Asn Arg Gln 225 230 235 240 ValArg Met Leu Ala Asp Leu Ser Leu Val Gly Cys Tyr Asn Leu Ser 245 250 255Phe Ile Pro Glu Gly Lys Arg Ala Gln Leu Leu Leu Glu Ser Ala Lys 260 265270 Lys Asn Leu Arg Gly Met Ala Phe Phe Gly Leu Thr Glu Phe Gln Arg 275280 285 Lys Thr Gln Tyr Leu Phe Glu Arg Thr Phe Asn Leu Lys Phe Ile Arg290 295 300 Pro Phe Met Gln Tyr Asn Ser Thr Arg Ala Gly Gly Val Glu ValAsp 305 310 315 320 Glu Asp Thr Ile Phe Phe Ile Glu Glu Leu Asn Asp LeuAsp Met Gln 325 330 335 Leu Tyr Asp Tyr Ala Lys Asp Leu Phe Gln Gln ArgTyr Gln Tyr Lys 340 345 350 Arg Gln Leu Glu Arg Arg Glu Gln Arg Leu ArgSer Arg Glu Glu Arg 355 360 365 Leu Leu His Arg Ala Lys Glu Ala Leu ProArg Glu Asp Ala Asp Glu 370 375 380 Pro Gly Arg Val Pro Thr Glu Asp TyrMet Ser His Ile Ile Glu Lys 385 390 395 400 Trp 4 465 PRT Homo sapiens 4Met Ala Ser Val Gly Asn Met Asp Glu Lys Ser Asn Lys Leu Leu Leu 1 5 1015 Ala Leu Val Met Leu Phe Leu Phe Ala Val Ile Val Leu Gln Tyr Val 20 2530 Cys Pro Gly Thr Glu Cys Gln Leu Leu Arg Leu Gln Ala Phe Ser Ser 35 4045 Pro Val Pro Asp Pro Tyr Arg Ser Glu Asp Glu Ser Ser Ala Arg Phe 50 5560 Val Pro Arg Tyr Asn Phe Thr Arg Gly Asp Leu Leu Arg Lys Val Asp 65 7075 80 Phe Asp Ile Lys Gly Asp Asp Leu Ile Val Phe Leu His Ile Gln Lys 8590 95 Thr Gly Gly Thr Thr Phe Gly Arg His Leu Val Arg Asn Ile Gln Leu100 105 110 Glu Gln Pro Cys Glu Cys Arg Val Gly Gln Lys Lys Cys Thr CysHis 115 120 125 Arg Pro Gly Lys Arg Glu Thr Trp Leu Phe Ser Arg Phe SerThr Gly 130 135 140 Trp Ser Cys Gly Leu His Ala Asp Trp Thr Glu Leu ThrSer Cys Val 145 150 155 160 Pro Ser Val Val Asp Gly Lys Arg Asp Ala ArgLeu Arg Pro Ser Arg 165 170 175 Asn Phe His Tyr Ile Thr Ile Leu Arg AspPro Val Ser Arg Tyr Leu 180 185 190 Ser Glu Trp Arg His Val Gln Arg GlyAla Thr Trp Lys Ala Ser Leu 195 200 205 His Val Cys Asp Gly Arg Pro ProThr Ser Glu Glu Leu Pro Ser Cys 210 215 220 Tyr Thr Gly Asp Asp Trp SerGly Cys Pro Leu Lys Glu Phe Met Asp 225 230 235 240 Cys Pro Tyr Asn LeuAla Asn Asn Arg Gln Val Arg Met Leu Ser Asp 245 250 255 Leu Thr Leu ValGly Cys Tyr Asn Leu Ser Val Met Pro Glu Lys Gln 260 265 270 Arg Asn LysVal Leu Leu Glu Ser Ala Lys Ser Asn Leu Lys His Met 275 280 285 Ala PhePhe Gly Leu Thr Glu Phe Gln Arg Lys Thr Gln Tyr Leu Phe 290 295 300 GluLys Thr Phe Asn Met Asn Phe Ile Ser Pro Phe Thr Gln Tyr Asn 305 310 315320 Thr Thr Arg Ala Ser Ser Val Glu Ile Asn Glu Glu Ile Gln Lys Arg 325330 335 Ile Glu Gly Leu Asn Phe Leu Asp Met Glu Leu Tyr Ser Tyr Ala Lys340 345 350 Asp Leu Phe Leu Gln Arg Tyr Gln Phe Met Arg Gln Lys Glu HisGln 355 360 365 Glu Ala Arg Arg Lys Arg Gln Glu Gln Arg Asp Phe Leu LysGly Arg 370 375 380 Leu Leu Gln Thr His Phe Gln Ser Gln Gly Gln Gly GlySer Gln Asn 385 390 395 400 Pro Asn Gln Asn Gln Ser Gln Asn Pro Asn ProAsn Ala Asn Gln Asn 405 410 415 Leu Thr Gln Asn Leu Met Gln Asn Leu ThrGln Ser Leu Ser Gln Lys 420 425 430 Glu Asn Arg Glu Ser Pro Lys Gln AsnSer Gly Lys Glu Gln Asn Asp 435 440 445 Asn Thr Ser Asn Gly Thr Asn AspTyr Ile Gly Ser Val Glu Lys Trp 450 455 460 Arg 465 5 504 PRT Homosapiens 5 Met Ala Ser Val Gly Asn Met Asp Glu Lys Ser Asn Lys Leu LeuLeu 1 5 10 15 Ala Leu Val Met Leu Phe Leu Phe Ala Val Ile Val Leu GlnTyr Val 20 25 30 Cys Pro Gly Thr Glu Cys Gln Leu Leu Arg Leu Gln Ala PheSer Ser 35 40 45 Pro Val Pro Asp Pro Tyr Arg Ser Glu Asp Glu Ser Ser AlaArg Phe 50 55 60 Val Pro Arg Tyr Asn Phe Thr Arg Gly Asp Leu Leu Arg LysVal Asp 65 70 75 80 Phe Asp Ile Lys Gly Asp Asp Leu Ile Val Phe Leu HisIle Gln Lys 85 90 95 Thr Gly Gly Thr Thr Phe Gly Arg His Leu Val Arg AsnIle Gln Leu 100 105 110 Glu Gln Pro Cys Glu Cys Arg Val Gly Gln Lys LysCys Thr Cys His 115 120 125 Arg Pro Gly Lys Arg Glu Thr Trp Leu Phe SerArg Phe Ser Thr Gly 130 135 140 Trp Ser Cys Gly Leu His Ala Asp Trp ThrGlu Leu Thr Ser Cys Val 145 150 155 160 Pro Ser Val Val Asp His Lys ArgAsp Ala Arg Leu Arg Pro Ser Arg 165 170 175 Trp Arg Ile Phe Gln Ile LeuAsp Ala Ala Ser Lys Asp Lys Arg Gly 180 185 190 Ser Ser Asn Thr Asn AlaGly Ala Asn Ser Pro Val Ser His Lys Asp 195 200 205 Pro Glu His Ile ArgVal Gly Asn Phe His Tyr Ile Thr Ile Leu Arg 210 215 220 Asp Pro Val SerArg Tyr Leu Ser Glu Trp Arg His Val Gln Arg Gly 225 230 235 240 Ala ThrTrp Lys Ala Ser Leu His Val Cys Asp Gly Arg Pro Pro Thr 245 250 255 SerGlu Glu Leu Pro Ser Cys Tyr Thr Gly Asp Asp Trp Ser Gly Cys 260 265 270Pro Leu Lys Glu Phe Met Asp Cys Pro Tyr Asn Leu Ala Asn Asn Arg 275 280285 Gln Val Arg Met Leu Ser Lys Leu Thr Leu Val Gly Cys Tyr Asn Leu 290295 300 Ser Val Met Pro Glu Lys Gln Arg Asn Lys Val Leu Leu Glu Ser Ala305 310 315 320 Lys Ser Asn Leu Lys His Met Ala Phe Phe Gly Leu Thr GluPhe Gln 325 330 335 Arg Lys Thr Gln Tyr Leu Phe Glu Lys Thr Phe Asn MetAsn Phe Ile 340 345 350 Ser Pro Phe Thr Gln Tyr Asn Thr Thr Arg Ala SerSer Val Glu Ile 355 360 365 Asn Glu Glu Ile Gln Lys Arg Ile Glu Gly LeuAsn Phe Leu Asp Met 370 375 380 Glu Leu Tyr Ser Tyr Ala Lys Asp Leu PheLeu Gln Arg Tyr Gln Phe 385 390 395 400 Met Arg Gln Lys Glu His Gln GluAla Arg Arg Lys Arg Gln Glu Gln 405 410 415 Arg Lys Phe Leu Lys Gly ArgLeu Leu Gln Thr His Phe Gln Ser Gln 420 425 430 Gly Gln Gly Gln Ser GlnAsn Pro Asn Gln Asn Gln Ser Gln Asn Pro 435 440 445 Asn Pro Asn Ala AsnGln Asn Leu Thr Gln Asn Leu Met Gln Asn Leu 450 455 460 Thr Gln Ser LeuSer Gln Lys Glu Asn Arg Glu Ser Pro Lys Gln Asn 465 470 475 480 Ser GlyLys Glu Gln Asn Asp Asn Thr Ser Asn Gly Thr Asn Asp Tyr 485 490 495 IleGly Ser Val Glu Lys Trp Arg 500 6 471 PRT Homo sapiens 6 Met Asp Glu ArgPhe Asn Lys Trp Leu Leu Thr Pro Val Leu Thr Leu 1 5 10 15 Leu Phe ValVal Ile Met Tyr Gln Tyr Val Ser Pro Ser Cys Thr Ser 20 25 30 Ser Cys ThrAsn Phe Gly Glu Gln Pro Arg Glu Gly Glu Ala Gly Pro 35 40 45 Pro Ala ValPro Gly Pro Ala Arg Arg Ala Gln Ala Pro Pro Glu Glu 50 55 60 Trp Glu ArgArg Pro Gln Leu Pro Pro Pro Pro Arg Gly Pro Pro Glu 65 70 75 80 Gly ProArg Gly Ala Ala Ala Pro Glu Glu Glu Asp Glu Glu Pro Gly 85 90 95 Asp ProArg Glu Gly Glu Glu Glu Glu Glu Glu Asp Glu Pro Asp Pro 100 105 110 GluAla Pro Glu Asn Gly Ser Leu Pro Arg Phe Val Pro Arg Phe Asn 115 120 125Phe Ser Leu Lys Ser Leu Thr Arg Phe Val Asp Phe Asn Ile Lys Gly 130 135140 Arg Asp Val Ile Val Phe Leu His Ile Gln Lys Thr Gly Gly Thr Thr 145150 155 160 Phe Gly Arg His Leu Val Lys Asn Ile Arg Leu Glu Gln Pro CysSer 165 170 175 Cys Lys Ala Gly Gln Lys Lys Cys Thr Cys His Arg Pro GlyLys Lys 180 185 190 Glu Thr Trp Leu Phe Ser Arg Phe Ser Thr Gly Trp SerCys Gly Leu 195 200 205 His Ala Asp Trp Thr Glu Leu Thr Asn Cys Val ProAla Ile Met Glu 210 215 220 Lys Lys Asp Cys Pro Arg Asn His Ser His ThrArg Asn Phe Tyr Tyr 225 230 235 240 Ile Thr Met Leu Arg Asp Pro Val SerArg Tyr Leu Ser Glu Trp Lys 245 250 255 His Val Gln Arg Gly Ala Thr TrpLys Thr Ser Leu His Met Cys Asp 260 265 270 Gly Arg Ser Pro Thr Pro AspGlu Leu Pro Thr Cys Tyr Pro Gly Asp 275 280 285 Asp Trp Ser Gly Val SerLeu Arg Glu Phe Met Asp Cys Thr Tyr Asn 290 295 300 Leu Ala Asn Asn ArgGln Val Arg Met Leu Ala Asp Ser Leu Ser Val 305 310 315 320 Gly Cys TyrAsn Leu Thr Phe Met Asn Glu Ser Glu Arg Asn Thr Ile 325 330 335 Leu LeuGln Ser Ala Lys Asn Asn Leu Lys Asn Met Ala Phe Phe Gly 340 345 350 LeuThr Glu Phe Gln Arg Lys Thr Gln Phe Leu Phe Glu Arg Thr Phe 355 360 365Asn Leu Lys Phe Ile Ser Pro Phe Thr Gln Phe Asn Ile Thr Arg Ala 370 375380 Ser Asn Val Glu Ile Asn Glu Gly Ala Arg Gln Arg Ile Glu Asp Leu 385390 395 400 Asn Phe Leu Asp Met Gln Leu Tyr Glu Tyr Ala Lys Asp Leu PheGln 405 410 415 Gln Arg Tyr His His Thr Lys Gln Leu Glu His Gln Arg AspArg Gln 420 425 430 Lys Arg Arg Glu Glu Arg Arg Leu Gln Arg Glu His ArgAsp His Gln 435 440 445 Trp Pro Lys Glu Asp Gly Ala Ala Glu Gly Thr ValThr Glu Asp Tyr 450 455 460 Asn Ser Gln Val Val Arg Trp 465 470

1. A method of transferring a sulfate on to the 6-O position of a GlcNAcsugar residue hi a polysaccharide preparation, the method comprising thesteps of (a) providing a polysaccharide preparation having GlcNAc sugarresidues, and (b) contacting the polysaccharide preparation with 6-OSTprotein in the presence of a sulfate donor under conditions which permitthe 6-OST protein to add a sulfate the 6-O-position of a GlcNAc sugarresidue.
 2. The method of claim 1, wherein the polysaccharidepreparation comprises heparan.
 3. The method of claim 1, wherein thepolysaccharide preparation comprises glucuronic acid (GlcUA) residues.4. A method as in claim 1, wherein the polysaccharide preparationincludes GlcUA—GlcNAc sugar residues.
 5. A method as in claim 1, whereinthe polysaccharide preparation includes disaccharides selected from theconsisting of GlcUA/IdoUA—GlcNS, IdoUA2S—GlcNS, and GlcUA—GlcNS3S.
 6. Amethod as in claim 1, wherein the polysaccharide preparation includesGlcNAc/NS6S—GlcUA—GlcNS3 S±6S—IdoUA2S—GlcNS6S.
 7. A method as in claim1, wherein the polysaccharide preparation includesGlcNAc/NS—GlcUA—GlcNS3 S±6S—IdoUA2S—GlcNS6S.
 8. A method as in claim 1,wherein the polysaccharide preparation includesGlcNAc/NS6S—GlcUA—GlcNS3S±—IdoUA2S—GlcNS6S.
 9. A method as in claim 1,wherein the polysaccharide preparation includes GlcNAc/NS6S—GlcUA—GlcNS3S±6S—IdoUA2S—GlcNS.
 10. The method of claim 1, wherein the 6-OST proteinis a recombinant protein.
 11. The method of claim 10, wherein the 6-OSTprotein is a human recombinant protein.
 12. The method of claim 10,wherein the recombinant protein is produced in a expression systemselected from the group consisting of baculovirus cells, yeast cells,bacterial cells, and mammalian cells.
 13. A method as in claim 1,wherein the sulfate donor is PAPS.
 14. A method as in claim 1, whereinthe 6-O-sulfation is performed in a reaction mixture comprising at leastone chloride salt, wherein the pH is between 6.5 and 7.5.
 15. The methodof claim 1, wherein the polysaccharide preparation is contacted with thepolysaccharide preparation with 6-OST protein in the presence of asulfate donor for at least 20 minutes.
 16. A method of enriching theportion of HS^(act) present in a polysaccharide preparation comprising:(a) providing a 3-O-sulfated polysaccharide preparation; and (b)contacting the preparation with 6-OST protein in the presence of asulfate donor under conditions, which permit the 6-OST protein to add asulfate the 6-O-position of a GlcNAc sugar residue, wherein, step (b)occurs concurrent with or subsequent to step (a).
 17. The method ofclaim 16, wherein the 3-O-sufated polysaccharide preparation is derivedfrom a cell that expresses 3-OST-1 protein.
 18. The method of claim 17,wherein the 3-O-sufated polysaccharide preparation is prepared bycontacting HS^(inact) with 3-OST-1 protein.
 19. The method of claim 16,wherein the polysaccharide preparation is derived from heparan.
 20. Themethod of claim 16, wherein the percentage of HS^(act) present in thepolysaccharide preparation following step (b) is greater than 70%. 21.The method of claim 16, wherein the percentage of HS^(act) present inthe polysaccharide preparation following step (b) is greater than 50%.22. The method of claim 16, wherein the polysaccharide preparationcomprise N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcUA)residues.
 23. A method as in claim 16, wherein the polysaccharidepreparation includes sugar residues selected from the group consistingof GlcUA/IdoUA—GlcNS, GlcUA—GlcNAc, IdoUA2S—GlcNS, and GlcUA—GlcNS3S.24. A method as in claim 16, wherein the sulfate donor comprises PAPS.25. A method as in claim 16, wherein the 6-O-sulfation is performed in areaction mixture comprising at least one chloride salt at a pH of about6.5-7.5.
 25. 26. A method as in any one of claim 1-25, wherein the 6-OSTprotein comprises a polypeptide selected from the group consisting of(a) SEQ ID NO. 3; (b) SEQ ID NO. 4; (c) SEQ ID NO. 5; (d) SEQ ID NO. 6;(e) an allelic variant of any of a-d; and (f) a functional fragment ofany one of a-d.
 27. A substantially pure preparation made by the methodof any one of claims 1-26.
 28. A mutant CHO cell that produces more than28% HS^(act), relative to HS^(total).
 29. A mutant CHO cell thatproduces between 28% and 50% HS^(act), relative to HS^(total).
 30. Themutant CHO cell of claim 28, produced by a method comprising: (a)transforming a CHO cell with multiple copies of 3-OST-1; (b)mutagenizing the cell obtained in step (a); (c) isolating a mutant cellfrom step (b) which fails to produce HS^(act); and (d) transforming thecell obtained in step (c) with 6-OST.
 31. A method of identifyingcomponents in a biosynthetic pathway comprising the steps of; a)providing a target cell which expresses at least the upstream componentsof the biosynthetic pathway; b) transforming the target cell withmultiple copies of an isolated biosynthetic pathway downstream gene; c)mutagenizing the transformed target cell; and d) identifying transformedand mutagenized target cells tat fail to express the phenotypecharacteristic of the biosynthetic pathway.
 32. The method of claim 31,further comprising step (e) correcting the step (d) cells, wherein thecorrected cells express the wild-type phenotype of the cell in step(a).33. The method of claim 32, wherein the correcting step comprisestransforming the cell with the nucleic acid that encodes an upstreamgene.
 34. The method of claim 33, wherein the upstream gene is a cDNA orgenomic DNA.
 35. The method of claim 31, wherein the cells of step (d)are transformed with a pool of preselected cDNAs for components of thebiosynthetic pathway.
 36. The method of claim 31, wherein the cells ofstep (d) are transformed with a cDNA library derived from a cell thatexpresses wild-type phenotype.
 37. The method of claim 32, wherein thecorrecting step comprises contacting the cells of step (d) with the geneproduct of an upstream gene.
 38. The method of claim 31, furthercomprising the step of isolating the cells from step (d).
 39. The methodof claim 31, further comprising the step of analyzing the cells of step(d).
 41. The method of claim 34, further comprising the step ofisolating the upstream gene in the biosynthetic pathway.
 42. The methodof claim 31, wherein the mutagenesis step comprises a mutagenesistechnique selected from the group consisting of chemical mutagenesis,ion radiation, and ultraviolet radiation.
 43. The method of claim 31,wherein the step of identifying the gene cDNA comprises complementationanalysis.
 44. The method of claim 31, wherein the identifying stepcomprises identifying the gene by a technique selected from the groupconsisting of Northern blot analysis, Southern blot analysis, andWestern blot analysis.
 45. The method of claim 31, wherein theidentifying further comprises isolating of the gene using PCR.